Novel chimpanzee erythropoietin (CHEPO) polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel chimpanzee erythropoietin polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, and antibodies which bind to the polypeptides of the present invention.

[0001] This is a continuation-in-part of copending application Ser. No.09/552,265 filed on Apr. 19, 2000, which is a continuation-in-part ofcopending application Ser. No. 09/307,307 filed on May 7, 1999.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the identification andisolation of novel chimpanzee erythropoietin polypeptides, nucleic acidmolecules encoding those polypeptides and to the recombinant productionof those polypeptides.

BACKGROUND OF THE INVENTION

[0003] Erythropoiesis, the production of red blood cells, occurscontinuously throughout the human life span to offset cell destruction.Erythropoiesis is a very precisely controlled physiological mechanismenabling sufficient numbers of red blood cells to be available in theblood for proper tissue oxygenation, but not so many that the cellswould impede circulation. The formation of red blood cells occurs in thebone marrow and is under control of the hormone, erythropoietin.

[0004] Erythropoietin, an acidic glycoprotein is approximately 34,000dalton molecular weight, may occur in three forms: alpha, beta andasialo. The alpha and beta forms different slightly in carbohydratecomponents have the same potency, biological activity and molecularweight. The asialo form is an alpha or beta form with the terminalcarbohydrate (sialic acid) removed. Erythropoietin is present in a verylow concentrations in plasma when the body is in a healthy state whereintissues receive sufficient oxygenation from the existing number oferythrocytes. This normal low concentration is enough to stimulatereplacement of red blood cells which are lost normally through aging.

[0005] The amount of erythropoietin in the circulation is increasedunder conditions of hypoxia when oxygen transport by blood cells in thecirculation is reduced. Hypoxia may be caused by loss of large amountsof blood through hemorrhage, destruction of red blood cells byover-exposure to radiation, reduction in oxygen intake due to highaltitudes or prolonged unconsciousness, or various forms of anemia. Inresponse to tissues undergoing hypoxic stress, erythropoietin willincrease red blood cell production by stimulating the conversion ofprimitive precursor cells in the bone marrow into proerythroblasts whichsubsequently mature, synthesize hemoglobin and are released into thecirculation as red blood cells. When the number of red blood cells incirculation is greater than needed for normal tissue oxygenrequirements, erythropoietin in circulation is decreased.

[0006] Because erythropoietin is essential in the process of red bloodcell formation, the hormone has potential useful application in both thediagnosis and treatment of blood disorders characterized by low ordefective red blood cell production. See, generally, Pennathur-Das, etal., Blood 63(5):1168-71 (1984) and Haddy, Am. Jour. Ped. Hematol.Oncol., 4:191-196 (1982) relating to erythropoietin in possibletherapies for sickle cell disease, and Eschbach et al., J. Clin. Invest.74(2):434-441 (1984), describing a therapeutic regimen for uremic sheepbased on in vivo response to erythropoietin-rich plasma infusions andproposing a dosage of 10 U EOP/kg per day for 15-40 days as correctiveof anemia of the type associated with chronic renal failure. See also,Krane, Henry Ford Hosp. Med. J., 31(3):177-181 (1983).

[0007] We describe herein the identification and characterization of anovel erythropoietin polypeptide derived from the chimpanzee, designatedherein as “CHEPO”.

SUMMARY OF THE INVENTION

[0008] A cDNA clone has been identified that has homology to nucleicacid encoding human erythropoietin that encodes a novel chimpanzeeerythropoietin polypeptide, designated in the present application as“CHEPO”.

[0009] In one embodiment, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes a CHEPOpolypeptide.

[0010] In one aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule encoding a CHEPO polypeptide having thesequence of amino acid residues from about 1 or about 28 to about 193,inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement of theDNA molecule of (a).

[0011] In another aspect, the isolated nucleic acid molecule comprises(a) a nucleotide sequence encoding a CHEPO polypeptide having thesequence of amino acid residues from about 1 or about 28 to about 193,inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement of thenucleotide sequence of (a).

[0012] In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule having the sequence of nucleotides fromabout 1 or about 82 to about 579, inclusive, of FIG. 2 (SEQ ID NO:3), or(b) the complement of the DNA molecule of (a).

[0013] In another aspect, the isolated nucleic acid molecule comprises(a) the nucleotide sequence of from about 1 or about 82 to about 579,inclusive, of FIG. 2 (SEQ ID NO:3), or (b) the complement of thenucleotide sequence of (a).

[0014] In another aspect, the invention concerns an isolated nucleicacid molecule which encodes an active CHEPO polypeptide as defined belowcomprising a nucleotide sequence that hybridizes to the complement of anucleic acid sequence that encodes amino acids 1 or about 28 to about193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5). Preferably,hybridization occurs under stringent hybridization and wash conditions.

[0015] In yet another aspect, the invention concerns an isolated nucleicacid molecule which encodes an active CHEPO polypeptide as defined belowcomprising a nucleotide sequence that hybridizes to the complement ofthe nucleic acid sequence between about nucleotides 1 or about 82 andabout 579, inclusive, of FIG. 2 (SEQ ID NO:3). Preferably, hybridizationoccurs under stringent hybridization and wash conditions.

[0016] In a further aspect, the invention concerns an isolated nucleicacid molecule which is produced by hybridizing a test DNA molecule understringent conditions with (a) a DNA molecule encoding a CHEPOpolypeptide having the sequence of amino acid residues from about 1 orabout 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b)the complement of the DNA molecule of (a), and, if the test DNA moleculehas at least about an 80% nucleic acid sequence identity, alternativelyat least about 81% nucleic acid sequence identity, alternatively atleast about 82% nucleic acid sequence identity, alternatively at leastabout 83% nucleic acid sequence identity, alternatively at least about84% nucleic acid sequence identity, alternatively at least about 85%nucleic acid sequence identity, alternatively at least about 86% nucleicacid sequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) or (b), and isolating the test DNA molecule.

[0017] In a specific aspect, the invention provides an isolated nucleicacid molecule comprising DNA encoding a CHEPO polypeptide without theN-terminal signal sequence and/or the initiating methionine, or iscomplementary to such encoding nucleic acid molecule. The signal peptidehas been tentatively identified as extending from about amino acidposition 1 to about amino acid position 27 in the sequence of FIG. 3(SEQ ID NOS:2 and 5). It is noted, however, that the C-terminal boundaryof the signal peptide may vary, but most likely by no more than about 5amino acids on either side of the signal peptide C-terminal boundary asinitially identified herein, wherein the C-terminal boundary of thesignal peptide may be identified pursuant to criteria routinely employedin the art for identifying that type of amino acid sequence element(e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al.,Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognizedthat, in some cases, cleavage of a signal sequence from a secretedpolypeptide is not entirely uniform, resulting in more than one secretedspecies. These polypeptides, and the polynucleotides encoding them, arecontemplated by the present invention. As such, for purposes of thepresent application, the signal peptide of the CHEPO polypeptide shownin FIG. 3 (SEQ ID NOS:2 and 5) extends from amino acids 1 to X of FIG. 3(SEQ ID NOS:2 and 5), wherein X is any amino acid from 23 to 32 of FIG.3 (SEQ ID NOS:2 and 5). Therefore, mature forms of the CHEPO polypeptidewhich are encompassed by the present invention include those comprisingamino acids X to 193 of FIG. 3 (SEQ ID NOS:2 and 5), wherein X is anyamino acid from 23 to 32 of FIG. 3 (SEQ ID NOS:2 and 5) and variantsthereof as described below. Isolated nucleic acid molecules encodingthese polypeptides are also contemplated.

[0018] Another embodiment is directed to fragments of a CHEPOpolypeptide coding sequence that may find use as, for example,hybridization probes or for encoding fragments of a CHEPO polypeptidethat may optionally encode a polypeptide comprising a binding site foran anti-CHEPO antibody. Such nucleic acid fragments are usually at leastabout 20 nucleotides in length, alternatively at least about 30nucleotides in length, alternatively at least about 40 nucleotides inlength, alternatively at least about 50 nucleotides in length,alternatively at least about 60 nucleotides in length, alternatively atleast about 70 nucleotides in length, alternatively at least about 80nucleotides in length, alternatively at least about 90 nucleotides inlength, alternatively at least about 100 nucleotides in length,alternatively at least about 110 nucleotides in length, alternatively atleast about 120 nucleotides in length, alternatively at least about 130nucleotides in length, alternatively at least about 140 nucleotides inlength, alternatively at least about 150 nucleotides in length,alternatively at least about 160 nucleotides in length, alternatively atleast about 170 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 190 nucleotides inlength, alternatively at least about 200 nucleotides in length,alternatively at least about 250 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 350nucleotides in length, alternatively at least about 400 nucleotides inlength, alternatively at least about 450 nucleotides in length,alternatively at least about 500 nucleotides in length, alternatively atleast about 600 nucleotides in length, alternatively at least about 700nucleotides in length, alternatively at least about 800 nucleotides inlength, alternatively at least about 900 nucleotides in length andalternatively at least about 1000 nucleotides in length, wherein in thiscontext the term “about” means the referenced nucleotide sequence lengthplus or minus 10% of that referenced length. In a preferred embodiment,the nucleotide sequence fragment is derived from any coding region ofthe nucleotide sequence shown in FIG. 1 (SEQ ID NO:1). It is noted thatnovel fragments of a CHEPO polypeptide-encoding nucleotide sequence maybe determined in a routine manner by aligning the CHEPOpolypeptide-encoding nucleotide sequence with other known nucleotidesequences using any of a number of well known sequence alignmentprograms and determining which CHEPO polypeptide-encoding nucleotidesequence fragment(s) are novel. All of such CHEPO polypeptide-encodingnucleotide sequences are contemplated herein and can be determinedwithout undue experimentation. Also contemplated are the CHEPOpolypeptide fragments encoded by these nucleotide molecule fragments,preferably those CHEPO polypeptide fragments that comprise a bindingsite for an anti-CHEPO antibody.

[0019] In another embodiment, the invention provides a vector comprisinga nucleotide sequence encoding CHEPO or its variants. The vector maycomprise any of the isolated nucleic acid molecules hereinaboveidentified.

[0020] A host cell comprising such a vector is also provided. By way ofexample, the host cells may be CHO cells, E. coli, or yeast. A processfor producing CHEPO polypeptides is further provided and comprisesculturing host cells under conditions suitable for expression of CHEPOand recovering CHEPO from the cell culture.

[0021] In another embodiment, the invention provides isolated CHEPOpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0022] In a specific aspect, the invention provides isolated nativesequence CHEPO polypeptide, which in certain embodiments, includes anamino acid sequence comprising residues from about 1 or about 28 toabout 193 of FIG. 3 (SEQ ID NOS:2 and 5).

[0023] In another aspect, the invention concerns an isolated CHEPOpolypeptide, comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to thesequence of amino acid residues from about 1 or about 28 to about 193,inclusive, of FIG. 3 (SEQ ID NOS:2 and 5).

[0024] In a specific aspect, the invention provides an isolated CHEPOpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as hereinbefore described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the CHEPO polypeptide and recovering the CHEPO polypeptidefrom the cell culture.

[0025] In yet another aspect, the invention concerns an isolated CHEPOpolypeptide, comprising the sequence of amino acid residues from about 1or about 28 to about 193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), ora fragment thereof which is biologically active or sufficient to providea binding site for an anti-CHEPO antibody, wherein the identification ofCHEPO polypeptide fragments that possess biological activity or providea binding site for an anti-CHEPO antibody may be accomplished in aroutine manner using techniques which are well known in the art.Preferably, the CHEPO fragment retains a qualitative biological activityof a native CHEPO polypeptide.

[0026] In a still further aspect, the invention provides a polypeptideproduced by (i) hybridizing a test DNA molecule under stringentconditions with (a) a DNA molecule encoding a CHEPO polypeptide havingthe sequence of amino acid residues from about 1 or about 28 to about193, inclusive, of FIG. 3 (SEQ ID NOS:2 and 5), or (b) the complement ofthe DNA molecule of (a), and if the test DNA molecule has at least aboutan 80% nucleic acid sequence identity, alternatively at least about 81%nucleic acid sequence identity, alternatively at least about 82% nucleicacid sequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) or (b), (ii) culturing a host cell comprisingthe test DNA molecule under conditions suitable for expression of thepolypeptide, and (iii) recovering the polypeptide from the cell culture.

[0027] In another embodiment, the invention provides chimeric moleculescomprising a CHEPO polypeptide fused to a heterologous polypeptide oramino acid sequence, wherein the CHEPO polypeptide may comprise anyCHEPO polypeptide, variant or fragment thereof as hereinbeforedescribed. An example of such a chimeric molecule comprises a CHEPOpolypeptide fused to an epitope tag sequence or a Fc region of animmunoglobulin.

[0028] In another embodiment, the invention provides an antibody asdefined below which specifically binds to a CHEPO polypeptide ashereinbefore described. Optionally, the antibody is a monoclonalantibody, an antibody fragment or a single chain antibody.

[0029] In yet another embodiment, the invention concerns agonists andantagonists of a native CHEPO polypeptide as defined below. In aparticular embodiment, the agonist or antagonist is an anti-CHEPOantibody or a small molecule.

[0030] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists to a CHEPO polypeptide whichcomprise contacting the CHEPO polypeptide with a candidate molecule andmonitoring a biological activity mediated by said CHEPO polypeptide.Preferably, the CHEPO polypeptide is a native CHEPO polypeptide.

[0031] In a still further embodiment, the invention concerns acomposition of matter comprising a CHEPO polypeptide, or an agonist orantagonist of a CHEPO polypeptide as herein described, or an anti-CHEPOantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

[0032] Another embodiment of the present invention is directed to theuse of a CHEPO polypeptide, or an agonist or antagonist thereof asherein described, or an anti-CHEPO antibody, for the preparation of amedicament useful in the treatment of a condition which is responsive tothe CHEPO polypeptide, an agonist or antagonist thereof or an anti-CHEPOantibody.

[0033] Yet another embodiment of the present invention is directed toCHEPO polypeptides having altered glycosylation patterns in one or moreregions of the polypeptide as compared to the native sequence CHEPOpolypeptide, preferably in the region surrounding and/or including aminoacid position 84 in the CHEPO amino acids sequence shown in FIG. 3 (SEQID NOS:2 and 5). In various embodiments, CHEPO variant polypeptides areprepared using well known techniques so as to create an N- or O-linkedglycosylation site at or near amino acid position 84 in the CHEPOpolypeptide sequence. For example, CHEPO polypeptides contemplated bythe present invention include those where (a) amino acids 81-84 of theCHEPO amino acid sequence shown in FIG. 3 (SEQ ID NOS:2 and 5) (i.e.,Met-Glu-Val-Arg; SEQ ID NO:6) are replaced by the amino acid sequenceAsn-X-Ser-X (SEQ ID NO:7) or Asn-X-Thr-X (SEQ ID NO:8), where X is anyamino acid except for Pro; (b) amino acids 82-85 of the CHEPO amino acidsequence shown in FIG. 3 (SEQ ID NOS:2 and 5) (i.e., Glu-Val-Arg-Gln;SEQ ID NO:9) are replaced by the amino acid sequence Asn-X-Ser-X (SEQ IDNO:7) or Asn-X-Thr-X (SEQ ID NO:8), where X is any amino acid except forPro; (c) amino acids 83-86 of the CHEPO amino acid sequence shown inFIG. 3 (SEQ ID NOS:2 and 5) (i.e., Val-Arg-Gln-Gln; SEQ ID NO:10) arereplaced by the amino acid sequence Asn-X-Ser-X (SEQ ID NO:7) orAsn-X-Thr-X (SEQ ID NO:8), where X is any amino acid except for Pro; or(d) amino acids 84-87 of the CHEPO amino acid sequence shown in FIG. 3(SEQ ID NOS:2 and 5) (i.e., Arg-Gln-Gln-Ala; SEQ ID NO:11) are replacedby the amino acid sequence Asn-X-Ser-X (SEQ ID NO:7) or Asn-X-Thr-X (SEQID NO:8), where X is any amino acid except for Pro, thereby creating anN-glycosylation site at those positions. Creating a glycosylation siteat the above described position(s) would be expected to result in amolecule that is less immunogenic in humans than the unmodifiedmolecule. Nucleic acids encoding these variant polypeptides are alsocontemplated herein as are vectors and host cells comprising thosenucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIGS. 1A-C show a nucleotide sequence (SEQ ID NO:1) of anisolated genomic DNA molecule containing a nucleotide sequence(nucleotides 134-146, 667-812, 1071-1157, 1760-1939 and 2074-2226,exclusive of others) encoding native sequence CHEPO. Also presented inthe genomic sequence are the locations of the start codon, exons andintrons as well as the amino acid sequence (SEQ ID NO:2) encoded by thecoding sequence of SEQ ID NO:1.

[0035]FIG. 2 shows the cDNA sequence of the CHEPO molecule (SEQ ID NO:3)and the amino acid sequence encoded thereby (SEQ ID NO:2).

[0036]FIG. 3 shows a comparison of the human erythropoietin amino acidsequence (human) (SEQ ID NO:4) and that of the chimp erythropoietin(chepo) described herein, wherein the amino acid designated “X” at aminoacid position 142 of the CHEPO sequence is either glutamine (SEQ IDNO:2) or lysine (SEQ ID NO:5).

[0037]FIG. 4 shows stimulation of proliferation of Ba/F3-EpoR cells byrecombinant human Epo (rhEPO, designated as “EPO” in the Figure) and aCHEPO-IgG1 immunoadhesin (“CHEPO-IgG”) as measured by the extent of[³H]-thymidine incorporation into the DNA.

[0038]FIG. 5 shows the effects of rhEPO (EPO) and CHEPO-IgG1 onproliferation of early and late cell markers (CD36 and CD71) oferythroid cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] I. Definitions

[0040] The terms “CHEPO polypeptide”, “CHEPO protein” and “CHEPO” whenused herein encompass native sequence CHEPO and CHEPO polypeptidevariants (which are further defined herein). The CHEPO polypeptide maybe isolated from a variety of sources, such as from human tissue typesor from another source, or prepared by recombinant and/or syntheticmethods.

[0041] A “native sequence CHEPO” comprises a polypeptide having the sameamino acid sequence as a CHEPO derived from nature. Such native sequenceCHEPO can be isolated from nature or can be produced by recombinantand/or synthetic means. The term “native sequence CHEPO” specificallyencompasses naturally-occurring truncated or secreted forms (e.g., anextracellular domain sequence), naturally-occurring variant forms (e.g.,alternatively spliced forms) and naturally-occurring allelic variants ofthe CHEPO. In one embodiment of the invention, the native sequence CHEPOis a mature or full-length native sequence CHEPO comprising amino acids1 to 193 of FIG. 3 (SEQ ID NOS:2 and 5). Also, while the CHEPOpolypeptides disclosed in FIG. 3 (SEQ ID NOS:2 and 5) is shown to beginwith the methionine residue designated herein as amino acid position 1,it is conceivable and possible that another methionine residue locatedeither upstream or downstream from amino acid position 1 in FIG. 3 (SEQID NOS:2 and 5) may be employed as the starting amino acid residue forthe CHEPO polypeptide.

[0042] “CHEPO variant polypeptide” means an active CHEPO polypeptide asdefined below having at least about 80% amino acid sequence identitywith the amino acid sequence of (a) residues 1 or about 28 to 193 of theCHEPO polypeptide shown in FIG. 3 (SEQ ID NOS:2 and 5), (b) X to 193 ofthe CHEPO polypeptide shown in FIG. 3 (SEQ ID NOS:2 and 5), wherein X isany amino acid residue from 23 to 32 of FIG. 3 (SEQ ID NOS:2 and 5) or(c) another specifically derived fragment of the amino acid sequenceshown in FIG. 3 (SEQ ID NOS:2 and 5). Such CHEPO variant polypeptidesinclude, for instance, CHEPO polypeptides wherein one or more amino acidresidues are added, or deleted, at the N- and/or C-terminus, as well aswithin one or more internal domains, of the sequence of FIG. 3 (SEQ IDNOS:2 and 5). Ordinarily, a CHEPO variant polypeptide will have at leastabout 80% amino acid sequence identity, alternatively at least about 81%amino acid sequence identity, alternatively at least about 82% aminoacid sequence identity, alternatively at least about 83% amino acidsequence identity, alternatively at least about 84% amino acid sequenceidentity, alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity with (a)residues 1 or about 28 to 193 of the CHEPO polypeptide shown in FIG. 3(SEQ ID NOS:2 and 5), (b) X to 193 of the CHEPO polypeptide shown inFIG. 3 (SEQ ID NOS:2 and 5), wherein X is any amino acid residue from 23to 32 of FIG. 3 (SEQ ID NOS:2 and 5) or (c) another specifically derivedfragment of the amino acid sequence shown in FIG. 3 (SEQ ID NOS:2 and5). CHEPO variant polypeptides do not encompass the native CHEPOpolypeptide sequence. Ordinarily, CHEPO variant polypeptides are atleast about 10 amino acids in length, alternatively at least about 20amino acids in length, alternatively at least about 30 amino acids inlength, alternatively at least about 40 amino acids in length,alternatively at least about 50 amino acids in length, alternatively atleast about 60 amino acids in length, alternatively at least about 70amino acids in length, alternatively at least about 80 amino acids inlength, alternatively at least about 90 amino acids in length,alternatively at least about 100 amino acids in length, alternatively atleast about 150 amino acids in length, alternatively at least about 200amino acids in length, alternatively at least about 300 amino acids inlength, or more.

[0043] “Percent (%) amino acid sequence identity” with respect to theCHEPO polypeptide sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a CHEPO sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2, whereinthe complete source code for the ALIGN-2 program is provided in Table 1below. The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc. and the source code shown in Table 1 has been filed withuser documentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1. The ALIGN-2 program should be compiled for use on a UNIXoperating system, preferably digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary.

[0044] For purposes herein, the % amino acid sequence identity of agiven amino acid sequence A to, with, or against a given amino acidsequence B (which can alternatively be phrased as a given amino acidsequence A that has or comprises a certain % amino acid sequenceidentity to, with, or against a given amino acid sequence B) iscalculated as follows:

100 times the fraction X/Y

[0045] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. As examples of % amino acid sequenceidentity calculations, Tables 2 and 3 below demonstrate how to calculatethe % amino acid sequence identity of the amino acid sequence designated“Comparison Protein” to the amino acid sequence designated “PRO”.

[0046] Unless specifically stated otherwise, all % amino acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % amino acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov or otherwise obtained from the NationalInstitute of Health, Bethesda, Md. NCBI-BLAST2 uses several searchparameters, wherein all of those search parameters are set to defaultvalues including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

[0047] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0048] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0049] “CHEPO variant polynucleotide” or “CHEPO variant nucleic acidsequence” means a nucleic acid molecule which encodes an active CHEPOpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with either (a) a nucleic acid sequence whichencodes residues 1 or about 28 to 193 of the CHEPO polypeptide shown inFIG. 3 (SEQ ID NOS:2 and 5), (b) a nucleic acid sequence which encodesresidues X to 193 of the CHEPO polypeptide shown in FIG. 3 (SEQ ID NOS:2and 5), wherein X is any amino acid residue from 23 to 32 of FIG. 3 (SEQID NOS:2 and 5) or (c) a nucleic acid sequence which encodes anotherspecifically derived fragment of the amino acid sequence shown in FIG. 3(SEQ ID NOS:2 and 5). Ordinarily, a CHEPO variant polynucleotide willhave at least about 80% nucleic acid sequence identity, alternatively atleast about 81% nucleic acid sequence identity, alternatively at leastabout 82% nucleic acid sequence identity, alternatively at least about83% nucleic acid sequence identity, alternatively at least about 84%nucleic acid sequence identity, alternatively at least about 85% nucleicacid sequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with either (a) a nucleic acid sequence which encodesresidues 1 or about 28 to 193 of the CHEPO polypeptide shown in FIG. 3(SEQ ID NOS:2 and 5), (b) a nucleic acid sequence which encodes residuesX to 193 of the CHEPO polypeptide shown in FIG. 3 (SEQ ID NOS:2 and 5),wherein X is any amino acid residue from 23 to 32 of FIG. 3 (SEQ IDNOS:2 and 5) or (c) a nucleic acid sequence which encodes anotherspecifically derived fragment of the amino acid sequence shown in FIG. 3(SEQ ID NOS:2 and 5). CHEPO polynucleotide variants do not encompass thenative CHEPO nucleotide sequence.

[0050] Ordinarily, CHEPO variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

[0051] “Percent (%) nucleic acid sequence identity” with respect to theCHEPO polypeptide-encoding nucleic acid sequences identified herein isdefined as the percentage of nucleotides in a candidate sequence thatare identical with the nucleotides in a CHEPO polypeptide-encodingnucleic acid sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full-length of the sequences being compared. For purposesherein, however, % nucleic acid sequence identity values are obtained asdescribed below by using the sequence comparison computer programALIGN-2, wherein the complete source code for the ALIGN-2 program isprovided in Table 1. The ALIGN-2 sequence comparison computer programwas authored by Genentech, Inc. and the source code shown in Table 1 hasbeen filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

[0052] For purposes herein, the % nucleic acid sequence identity of agiven nucleic acid sequence C to, with, or against a given nucleic acidsequence D (which can alternatively be phrased as a given nucleic acidsequence C that has or comprises a certain % nucleic acid sequenceidentity to, with, or against a given nucleic acid sequence D) iscalculated as follows:

100 times the fraction W/Z

[0053] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5 below demonstrate how to calculate the %nucleic acid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”.

[0054] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described above using theALIGN-2 sequence comparison computer program. However, % nucleic acidsequence identity may also be determined using the sequence comparisonprogram NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389-3402(1997)). The NCBI-BLAST2 sequence comparison program may be downloadedfrom http://www.ncbi.nlm.nih.gov or otherwise obtained from the NationalInstitute of Health, Bethesda, Md. NCBI-BLAST2 uses several searchparameters, wherein all of those search parameters are set to defaultvalues including, for example, unmask=yes, strand=all, expectedoccurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

[0055] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0056] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0057] In other embodiments, CHEPO variant polynucleotides are nucleicacid molecules that encode an active CHEPO polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding the full-length CHEPOpolypeptide shown in FIG. 3 (SEQ ID NOS:2 and 5). CHEPO variantpolypeptides may be those that are encoded by a CHEPO variantpolynucleotide.

[0058] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Preferably, the isolated polypeptide is free of association with allcomponents with which it is naturally associated. Contaminant componentsof its natural environment are materials that would typically interferewith diagnostic or therapeutic uses for the polypeptide, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the polypeptide will be purified (1) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (2)to homogeneity by SDS-PAGE under non-reducing or reducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated polypeptideincludes polypeptide in situ within recombinant cells, since at leastone component of the CHEPO natural environment will not be present.Ordinarily, however, isolated polypeptide will be prepared by at leastone purification step.

[0059] An “isolated” nucleic acid molecule encoding a CHEPO polypeptideis a nucleic acid molecule that is identified and separated from atleast one contaminant nucleic acid molecule with which it is ordinarilyassociated in the natural source of the CHEPO-encoding nucleic acid.Preferably, the isolated nucleic is free of association with allcomponents with which it is naturally associated. An isolatedCHEPO-encoding nucleic acid molecule is other than in the form orsetting in which it is found in nature. Isolated nucleic acid moleculestherefore are distinguished from the CHEPO-encoding nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule encoding a CHEPO polypeptide includes CHEPO-encodingnucleic acid molecules contained in cells that ordinarily express CHEPOwhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

[0060] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0061] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0062] The term “antibody” is used in the broadest sense andspecifically covers, for example, single anti-CHEPO monoclonalantibodies (including agonist, antagonist, and neutralizing antibodies),anti-CHEPO antibody compositions with polyepitopic specificity, singlechain anti-CHEPO antibodies, and fragments of anti-CHEPO antibodies (seebelow). The term “monoclonal antibody” as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts.

[0063] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0064] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0065] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and%SDS) less stringent that those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1×SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

[0066] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a CHEPO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

[0067] As used herein, the term “imumunoadhesin” designatesantibody-like molecules which combine the binding specificity of aheterologous protein (an “adhesin”) with the effector functions ofimmunoglobulin constant domains. Structurally, the immunoadhesinscomprise a fusion of an amino acid sequence with the desired bindingspecificity which is other than the antigen recognition and binding siteof an antibody (i.e., is “heterologous”), and an immunoglobulin constantdomain sequence. The adhesin part of an immunoadhesin molecule typicallyis a contiguous amino acid sequence comprising at least the binding siteof a receptor or a ligand. The immunoglobulin constant domain sequencein the immunoadhesin may be obtained from any immunoglobulin, such asIgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2),IgE, IgD or IgM.

[0068] “Active” or “activity” for the purposes herein refers to form(s)of CHEPO which retain a biological and/or an immunological activity ofnative or naturally-occurring CHEPO, wherein “biological” activityrefers to a biological function (either inhibitory or stimulatory)caused by a native or naturally-occurring CHEPO other than the abilityto induce the production of an antibody against an antigenic epitopepossessed by a native or naturally-occurring CHEPO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring CHEPO. Preferred biological activities includes, forexample, the ability to regulate red blood cell production, to bind toreceptors on the surface of committed progenitor cells of the bonemarrow and/or other hematopoietic tissues and/or to induce proliferationand/or terminal maturation of erythroid cells.

[0069] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native CHEPO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a nativeCHEPO polypeptide disclosed herein. Suitable agonist or antagonistmolecules specifically include agonist or antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativeCHEPO polypeptides, peptides, small organic molecules, etc. Methods foridentifying agonists or antagonists of a CHEPO polypeptide may comprisecontacting a CHEPO polypeptide with a candidate agonist or antagonistmolecule and measuring a detectable change in one or more biologicalactivities normally associated with the CHEPO polypeptide.

[0070] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

[0071] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

[0072] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0073] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0074] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0075] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062 [1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0076] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen.

[0077] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0078] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fabfragments differ from Fab′ fragments by the addition of a few residuesat the carboxy terminus of the heavy chain CH1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

[0079] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains.

[0080] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

[0081] “Single-chain Fv” or “sFv” antibody fragments comprise the VH andVL domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the sFvto form the desired structure for antigen binding. For a review of sFv,see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0082] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993).

[0083] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0084] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

[0085] An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

[0086] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0087] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as a CHEPO polypeptide or antibody thereto) to a mammal.The components of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.

[0088] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons.

[0089] An “effective amount” of a polypeptide disclosed herein or anagonist or antagonist thereof is an amount sufficient to carry out aspecifically stated purpose. An “effective amount” may be determinedempirically and in a routine manner, in relation to the stated purpose.TABLE 1 /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /*  A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ {2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1, −2, −1, 0, _M, 1, 0,−2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ {0, 3, −4, 3, 2, −5, 0, 1, −2, 0,0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0, −3, 1}, /* C */ {−2, −4,15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4, _M, −3, −5, −4, 0, −2, 0,−2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3, −6, 1, 1, −2, 0, 0, −4, −3,2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4, 2}, /* E */ {0, 2, −5, 3, 4,−5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4,3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2, 1, 0, −5, 2, 0, −4, _M, −5,−5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */ {1, 0, −3, 1, 0, −5, 5, −2,−3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0, 0, −1, −7, 0, −5, 0}, /* H*/ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2, −2, 2, _M, 0, 3, 2, −1,−1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2, −2, −2, 1, −3, −2, 5, 0,−2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5, 0, −1, −2}, /* J */ {0,0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0, 5, −3, 0, 1, _M, −1, 1,3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3, −6, −4, −3, 2, −4, −2,2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0, 2, −2, 0, −1, −2} /* M*/ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6, −2, _M, −2, −1, 0, −2,−1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2, 1, −4, 0, 2, −2, 0, 1,−3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,} /* P */ {1, −1, −3, −1, −1, −5, −1,0, −2, 0, −1, −3, −2, −1, _M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /* Q*/ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1, −1,0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0, 3,−3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ {1, 0, 0, 0,0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0, −3,0}, /* T */ {1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0, −1,−1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2, −2,−2, −1, −1, −2, 4, 0, −2, 2, 2, −2, _M, −1, −2, −2, −1, 0, 0, 4, −6, 0,−2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4, −4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ {0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1, _M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4}, }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2 {circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2];   /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1< <(‘D’-‘A’))|(1< <(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0×FFFFFFF, 1< <10, 1< <11, 1< <12, 1< <13, 1< <14, 1< <15, 1<<16, 1< <17, 1< <18, 1< <19, 1< <20, 1< <21, 1< <22, 1< <23, 1< <24, 1<<25|(1< <(‘E’-‘A’))|(1< <(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if (ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py;   /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy < = len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy < = len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx < =len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx = = 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0]−ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx = 0;} ...nw for (py = seqx[1], yy = 1; yy < = len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 > =dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) > =dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 > =delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis > = delx && mis > = dely[yy])col1[yy] = mis; else if (delx > = dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx > = MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij > = MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] > = MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij > = MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx = = len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }(void) free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print() -- only routinevisible outside this module  *  * static:  * getmat() -- trace back bestpath, count matches: print()  * pr_align() -- print alignment ofdescribed in array p[]: print()  * dumpblock() -- dump a block of lineswith numbers, stars: pr_align()  * nums() -- put out a number line:dumpblock()  * putline() -- put out a line (name, [num], seq, [num]):dumpblock()  * stars() - -put a line of stars: dumpblock()  *stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) = = 0) { fprintf(stderr, “%s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ = = pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (n1++ = = pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm = = 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”:“residue”, (ngapx = = 1)? “”:“s”); fprintf(fx, “%s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “ (%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy = =1)? “”:“s”); fprintf(fx,“%s”, outx); } if (dna) fprintf(fx, “\n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap= = 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap = = 1)?“” : “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2; i++) { nn = stripname(namex[i]); if(nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] = = pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] = =pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn = =olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for (i= 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) !=‘ ’)) { if (i = = 0) nums(i); if (i = = 0 && *out[1]) stars();putline(i); if (i = = 0 && *out[1]) fprintf(fx, star); if (i = = 1)nums(i); } } } /*  * put out a number line: dumpblock()  */ staticnums(ix) nums int  ix; /* index in out[] holding seq line */ { charnline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn =nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py= out[ix]; *py; py++, pn++) { if (*py = = ‘ ’ || *py = = ‘−’) *pn = ‘ ’;else { if (i%10 = = 0 || (i = = 1 && nc[ix] != 1)) { j = (i < 0)? −i :i; for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px =‘−’; } else *pn = ‘ ’; i++; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline;*pn; pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put outa line (name, [num], seq, [num]): dumpblock()  */ static putline(ix)putline int   ix; { ...putline int i; register char *px; for (px =namex[ix], i = 0; *px && *px != ‘:’; px++, i++) (void) putc(*px, fx);for (; i < lmax+P_SPC; i++) (void) putc(‘ ’, fx); /* these count from 1: * ni[] is current element (from 1)  * nc[] is number at start ofcurrent line  */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F,fx); (void) putc(‘\n’, fx); } /*  * put a line of stars (seqs always inout[0], out[1]): dumpblock()  */ static stars() stars { int i; registerchar *p0, *p1, cx, *px; if (!*out[0] || (*out[0] = = ‘ ’ && *(po[0]) = =‘ ’) || !*out[1] || (*out[1] = = ‘ ’ && *(po[1]) = = ‘ ’)) return; px =star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for (p0 = out[0], p1 =out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) && isalpha(*p1)) { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } else if (!dna &&_day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } else cx = ‘ ’;*px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path or prefixfrom pn, return len: pr_align()  */ static stripname(pn) stripname char*pn; /* file name (may be path) */ { register char *px, *py; py = 0; for(px = pn; *px; px++) if (*px = = ‘/’) py = px + 1; if (py) (void)strcpy(pn, py); return(strlen(pn)); } /*  * cleanup() -- cleanup any tmpfile  * getseq() -- read in seq, set dna, len, maxlen  * g_calloc() --calloc() with error checkin  * readjmps() -- get the good jmps, from tmpfile if necessary  * writejmps() -- write a filled array of jmps to atmp file: nw()  */ #include “nw.h” #include <sys/file.h> char *jname =“/tmp/homgXXXXXX”; /* tmp file for jmps */ FILE *fj; int cleanup(); /*cleanup tmp file */ long lseek(); /*  * remove any tmp file if we blow */ cleanup(i) cleanup int i; { if (fj) (void) unlink(jname); exit(i); }/*  * read, return ptr to seq, set dna, len, maxlen  * skip linesstarting with ‘;’, ‘<’, or ‘>’  * seq in upper or lower case  */ char  *getseq(file, len) getseq char *file; /* file name */ int *len; /* seqlen */ { char line[1024], *pseq; register char *px, *py; int natgc,tlen; FILE *fp; if ((fp = fopen(file, “r”)) = = 0) { fprintf(stderr,“%s: can't read %s\n”, prog, file); exit(1); } tlen = natgc = 0; while(fgets(line, 1024, fp)) { if (*line = = ‘;’ || *line = = ‘<’ || *line= = ‘>’) continue; for (px = line; *px != ‘\n’; px++) if (isupper(*px)|| islower(*px)) tlen++; } if ((pseq = malloc((unsigned)(tlen+6))) = =0) { fprintf(stderr, “%s: malloc() failed to get %d bytes for %s\n”,prog, tlen+6, file); exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] =‘\0’; ...getseq py = pseq + 4; *len = tlen; rewind(fp); while(fgets(line, 1024, fp)) { if (*line = = ‘;’ || *line = = ‘<’ || *line= = ‘>’) continue; for (px = line; *px != ‘\n’; px++) { if(isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ = toupper(*px);if (index(“ATGCU”, *(py−1))) natgc++; } } *py++ = ‘\0’; *py = ‘\0’;(void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); } char *g_calloc(msg, nx, sz) g_calloc char *msg; /* program, calling routine */int nx, sz; /* number and size of elements */ { char *px, *calloc(); if((px = calloc((unsigned)nx, (unsigned)sz)) = = 0) { if (*msg) {fprintf(stderr, “%s: g_calloc() failed %s (n= %d, sz= %d)\n”, prog, msg,nx, sz); exit(1); } } return(px); } /*  * get final jmps from dx[] ortmp file, set pp[], reset dmax: main()  */ readjmps() readjmps { int fd= −1; int siz, i0, i1; register i, j, xx; if (fj) { (void) fclose(fj);if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr, “%s: can'topen() %s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1 = 0, dmax0= dmax, xx = len0; ; i++) { while (1) { for (j = dx[dmax].ijmp; j > = 0&& dx[dmax].jp.x[j] > = xx; j−−) ; ...readjmps if (j < 0 &&dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0); (void)read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void) read(fd,(char *)&dx[dmax].offset, sizeof(dx[dmax].offset)); dx[dmax].ijmp =MAXJMP−1; } else break; } if (i >= JMPS) { fprintf(stderr, “%s: too manygaps in alignment\n”, prog); cleanup(1); } if (j > = 0) { siz =dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax += siz; if (siz < 0) { /*gap in second seq */ pp[1].n[i1] = −siz; xx += siz; /* id = xx − yy +len1 − 1  */ pp[1].x[i1] = xx − dmax + len1 − 1; gapy++; ngapy −= siz;/* ignore MAXGAP when doing endgaps */ siz = (−siz < MAXGAP || endgaps)?−siz : MAXGAP; i1++; } else if (siz > 0) { /* gap in first seq */pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx += siz; /* ignoreMAXGAP when doing endgaps */ siz = (siz < MAXGAP || endgaps)? siz :MAXGAP; i0++; } } else break; } /* reverse the order of jmps  */ for (j= 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j]; pp[0].n[j] =pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0];pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i =pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd > = 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw()  */writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp() %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) = = 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

[0090] TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein

[0091] TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein

[0092] TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA

[0093] TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length = 9 nucleotides)

[0094] II. Compositions and Methods of the Invention

[0095] A. Full-length CHEPO Polypeptide

[0096] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as CHEPO. In particular, DNA encoding a CHEPO polypeptidehas been identified and isolated, as disclosed in further detail in theExamples below.

[0097] B. CHEPO Variants

[0098] In addition to the full-length native sequence CHEPO polypeptidesdescribed herein, it is contemplated that CHEPO variants can beprepared. CHEPO variants can be prepared by introducing appropriatenucleotide changes into the CHEPO DNA, and/or by synthesis of thedesired CHEPO polypeptide. Those skilled in the art will appreciate thatamino acid changes may alter post-translational processes of the CHEPO,such as changing the number or position of glycosylation sites oraltering the membrane anchoring characteristics.

[0099] Variations in the native full-length sequence CHEPO or in variousdomains of the CHEPO described herein, can be made, for example, usingany of the techniques and guidelines for conservative andnon-conservative mutations set forth, for instance, in U.S. Pat. No.5,364,934. Variations may be a substitution, deletion or insertion ofone or more codons encoding the CHEPO that results in a change in theamino acid sequence of the CHEPO as compared with the native sequenceCHEPO. Optionally the variation is by substitution of at least one aminoacid with any other amino acid in one or more of the domains of theCHEPO. Guidance in determining which amino acid residue may be inserted,substituted or deleted without adversely affecting the desired activitymay be found by comparing the sequence of the CHEPO with that ofhomologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

[0100] CHEPO polypeptide fragments are provided herein. Such fragmentsmay be truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the CHEPO polypeptide.

[0101] CHEPO fragments may be prepared by any of a number ofconventional techniques. Desired peptide fragments may be chemicallysynthesized. An alternative approach involves generating CHEPO fragmentsby enzymatic digestion, e.g., by treating the protein with an enzymeknown to cleave proteins at sites defined by particular amino acidresidues, or by digesting the DNA with suitable restriction enzymes andisolating the desired fragment. Yet another suitable technique involvesisolating and amplifying a DNA fragment encoding a desired polypeptidefragment, by polymerase chain reaction (PCR). Oligonucleotides thatdefine the desired termini of the DNA fragment are employed at the 5′and 3′ primers in the PCR. Preferably, CHEPO polypeptide fragments shareat least one biological and/or immunological activity with the nativeCHEPO polypeptides shown in FIG. 3 (SEQ ID NOS:2 and 5).

[0102] In particular embodiments, conservative substitutions of interestare shown in Table 6 under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leunorleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrleu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu ala;norleucine

[0103] Substantial modifications in function or immunological identityof the CHEPO polypeptide are accomplished by selecting substitutionsthat differ significantly in their effect on maintaining (a) thestructure of the polypeptide backbone in the area of the substitution,for example, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain. Naturally occurring residues are divided into groupsbased on common side-chain properties:

[0104] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0105] (2) neutral hydrophilic: cys, ser, thr;

[0106] (3) acidic: asp, glu;

[0107] (4) basic: asn, gin, his, lys, arg;

[0108] (5) residues that influence chain orientation: gly, pro; and

[0109] (6) aromatic: trp, tyr, phe.

[0110] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0111] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the CHEPO variant DNA.

[0112] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

[0113] C. Modifications of CHEPO

[0114] Covalent modifications of CHEPO are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a CHEPO polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the CHEPO. Derivatization withbifunctional agents is useful, for instance, for crosslinking CHEPO to awater-insoluble support matrix or surface for use in the method forpurifying anti-CHEPO antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0115] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains [T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0116] Another type of covalent modification of the CHEPO polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence CHEPO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequenceCHEPO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins, involving a change in the natureand proportions of the various carbohydrate moieties present.

[0117] Addition of glycosylation sites to the CHEPO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence CHEPO (for O-linkedglycosylation sites). The CHEPO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the CHEPO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

[0118] Another means of increasing the number of carbohydrate moietieson the CHEPO polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0119] Removal of carbohydrate moieties present on the CHEPO polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0120] Another type of covalent modification of CHEPO comprises linkingthe CHEPO polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0121] The CHEPO of the present invention may also be modified in a wayto form a chimeric molecule comprising CHEPO fused to another,heterologous polypeptide or amino acid sequence.

[0122] In one embodiment, such a chimeric molecule comprises a fusion ofthe CHEPO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the CHEPO. The presence ofsuch epitope-tagged forms of the CHEPO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe CHEPO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0123] In an alternative embodiment, the chimeric molecule may comprisea fusion of the CHEPO with an immunoglobulin or a particular region ofan immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an immunoadhesin), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a CHEPO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgG1 molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0124] The CHEPO-immunoadhesins are preferably produced by expression ina recombinant host cell, ans isolation therefrom. Specifically, toobtain expression of a CHEPO-immunoadhesin, one or more expressionvector(s) is/are introduced into host cells by transformation ortransfection and the resulting recombinant host cells are cultured inconventional nutrient media, modified as appropriate for inducingpromoters, selecting recombinant cells, and/or amplifying CHEPO-Ig DNA.The following description illustrates the recombinant production ofCHEPO and CHEPO-immunoadhesins, also referred to as CHEPO-Ig.

[0125] D. Preparation of CHEPO and CHEPO-immunoadhesins.

[0126] The description below relates primarily to production of CHEPOand CHEPO-immunoadhesins by culturing cells transformed or transfectedwith a vector containing CHEPO or the corresponding CHEPO-Ig nucleicacid. It is, of course, contemplated that alternative methods, which arewell known in the art, may be employed to prepare CHEPO andCHEPO-immunoadhesins. For instance, the CHEPO or CHEPO-Ig sequence, orportions thereof, may be produced by direct peptide synthesis usingsolid-phase techniques [see, e.g., Stewart et al., Solid-Phase PeptideSynthesis, W. H. Freeman Co., San Francisco, Calif. (1969); Merrifield,J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro protein synthesis maybe performed using manual techniques or by automation. Automatedsynthesis may be accomplished, for instance, using an Applied BiosystemsPeptide Synthesizer (Foster City, Calif.) using manufacturer'sinstructions Various portions of CHEPO and immunoadhesin may bechemically synthesized separately and combined using chemical orenzymatic methods to produce the desired full-length CHEPO orCHEPO-immunoadhesin.

[0127] 1. Isolation of DNA Encoding CHEPO

[0128] DNA encoding CHEPO may be obtained from a cDNA library preparedfrom tissue believed to possess CHEPO mRNA and to express it at adetectable level. Accordingly, human CHEPO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The CHEPO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

[0129] Libraries can be screened with probes (such as antibodies toCHEPO or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic with the selected probe may be conducted usingstandard procedures, such as described in Sambrook et al., MolecularCloning: A Laboratory Manual (New York: Cold Spring Harbor LaboratoryPress, 1989). An alternative means to isolate the gene encoding CHEPO isto use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCRPrimer: A Laboratory Manual (Cold Spring Harbor Laboratory Press,1995)].

[0130] The Examples below described techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0131] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined using methods known in the art and as described herein.

[0132] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0133] 2. Construction of Nucleic Acid Encoding CHEPO-Immunoadhesins

[0134] DNA encoding immunoglobulin light or heavy chain constant regionsis known or readily available from cDNA libraries or is synthesized. Seefor example, Adams et al., Biochemistry, 19:2711-2719 (1980); Gough etal., Biochemistry, 19:2702-2710 (1980); Dolby et al., P.N.A.S. USA,77:6027-6031 (1980); Rice et al., P.N.A.S. USA, 79:7862-7865 (1982);Falkner et al., Nature, 298:286-288 (1982); and Morrison et al., Ann.Rev. Immunol., 2:239-256 (1984).

[0135] When preparing the immunoadhesins of the present invention,preferably nucleic acid encoding an extracellular domain of a naturalreceptor is fused C-terminally to nucleic acid encoding the N-terminusof an immunoglobulin constant domain sequence, however N-terminalfusions are also possible. Typically, in such fusions the encodedchimeric polypeptide will retain at least functionally active hinge, CH2and CH3 domains of the constant region of an immunoglobulin heavy chain.Fusions are also made to the C-terminus of the Fc portion of a constantdomain, or immediately N-terminal to the CH1 of the heavy chain or thecorresponding region of the light chain. The resultant DNA fusionconstruct is expressed in appropriate host cells.

[0136] Nucleic acid molecules encoding amino acid sequence variants ofnative sequence extracellular domains (such as from CHEPO) and/or theantibody sequences used to prepare the desired immunoadhesin, areprepared by a variety of methods known in the art. These methodsinclude, but are not limited to, isolation from a natural source (in thecase of naturally occurring amino acid sequence variants, such as thosementioned above in connection with CHEPO) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, and cassette mutagenesis of an earlier prepared variant ora non-variant version of native sequence CHEPO.

[0137] Variations in the native sequence as described above can be madeusing any of the techniques and guidelines for conservative andnon-conservative mutations set forth in Table 6 above.

[0138] In a preferred embodiment, the nucleic acid encodes a chimericmolecule in which the CHEPO sequence is fused to the N-terminus of theC-terminal portion of an antibody (in particular the Fc domain),containing the effector functions of an immunoglobulin, e.g. IgG1. It ispossible to fuse the entire heavy chain constant region to the CHEPOsequence. However, more preferably, a sequence beginning in the hingeregion just upstream of the papain cleavage site (which defines IgG Fcchemically; residue 216, taking the first residue of heavy chainconstant region to be 114 [Kobet et al., supra], or analogous sites ofother immunoglobulins) is used in the fusion. In a particularlypreferred embodiment, the CHEPO sequence is fused to the hinge regionand CH2 and CH3 or CH1, hinge, CH2 and CH3 domains of an IgG1, IgG2, orIgG3 heavy chain. The precise site at which the fusion is made is notcritical, and the optimal site can be determined by routineexperimentation.

[0139] For human immunoadhesins, the use of human IgG1 and IgG3immunoglobulin sequences is preferred. A major advantage of using IgG1is that IgG1 immunoadhesins can be purified efficiently on immobilizedprotein A. In contrast, purification of IgG3 requires protein G, asignificantly less versatile medium. However, other structural andfunctional properties of immunoglobulins should be considered whenchoosing the Ig fusion partner for a particular immunoadhesinconstruction. For example, the IgG3 hinge is longer and more flexible,so it can accommodate larger “adhesin” domains that may not fold orfunction properly when fused to IgG1. Another consideration may bevalency; IgG immunoadhesins are bivalent homodimers, whereas Ig subtypeslike IgA and IgM may give rise to dimeric or pentameric structures,respectively, of the basic Ig homodimer unit.

[0140] For CHEPO-Ig immunoadhesins designed for in vivo application, thepharmacokinetic properties and the effector functions specified by theFc region are important as well. Although IgG1, IgG2 and IgG4 all havein vivo half-lives of 21 days, their relative potencies at activatingthe complement system are different. IgG4 does not activate complement,and IgG2 is significantly weaker at complement activation than IgG1.Moreover, unlike IgG1, IgG2 does not bind to Fc receptors on mononuclearcells or neutrophils. While IgG3 is optimal for complement activation,its in vivo half-life is approximately one third of the other IgGisotypes.

[0141] Another important consideration for immunoadhesins designed to beused as human therapeutics is the number of allotypic variants of theparticular isotype. In general, IgG isotypes with fewerserologically-defined allotypes are preferred. For example, IgG1 hasonly four serologically-defined allotypic sites, two of which (G1m and2) are located in the Fc region; and one of these sites G1m1, isnon-immunogenic. In contrast, there are 12 serologically-definedallotypes in IgG3, all of which are in the Fc region; only three ofthese sites (G3m5, 11 and 21) have one allotype which is nonimmunogenic.Thus, the potential immunogenicity of an IgG3 immunoadhesin is greaterthan that of an IgG1 immunoadhesin.

[0142] The cDNAs encoding the CHEPO sequence and the Ig parts of theimmunoadhesin are inserted in tandem into a plasmid vector that directsefficient expression in the chosen host cells. For expression inmammalian cells pRK5-based vectors [Schall et al., Cell 61, 361-370(1990)] and CDM8-based vectors [Seed, Nature 329, 840 (1989)] may, forexample, be used. The exact junction can be created by removing theextra sequences between the designed junction codons usingoligonucleotide-directed deletional mutagenesis [Zoller and Smith,Nucleic Acids Res. 10, 6487 (1982); Capon et al., Nature 337, 525-531(1989)]. Synthetic oligonucleotides can be used, in which each half iscomplementary to the sequence on either side of the desired junction;ideally, these are 36 to 48-mers. Alternatively, PCR techniques can beused to join the two parts of the molecule in-frame with an appropriatevector.

[0143] Although the presence of an immunoglobulin light chain is notrequired in the immunoadhesins of the present invention, animmunoglobulin light chain might be present either covalently associatedto CHEPO-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the CHEPO sequence. In the former case, DNA encoding animmunoglobulin light chain is typically coexpressed with the DNAencoding the CHEPO-immunoglobulin heavy chain fusion protein. Uponsecretion, the hybrid heavy chain and the light chain will be covalentlyassociated to provide an immunoglobulin-like structure comprising twodisulfide-linked immunoglobulin heavy chain-light chain pairs. Methodsuitable for the preparation of such structures are, for example,disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

[0144] 3. Selection and Transformation of Host Cells

[0145] The following procedure for the selection and transformation ofhost cells are equally applicable to the recombinant production of CHEPOand CHEPO-immunoadhesins. Host cells are transfected or transformed withexpression or cloning vectors described herein for CHEPO production andcultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences. The culture conditions, such as media,temperature, pH and the like, can be selected by the skilled artisanwithout undue experimentation. In general, principles, protocols, andpractical techniques for maximizing the productivity of cell culturescan be found in Mammalian Cell Biotechnology: a Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[0146] Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0147] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli , Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA ; E. coli W3110 strain 9E4, whichhas the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan^(r) ; E. coli W3110 strain 37D6, which hasthe complete genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT rbs7ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6 with anon-kanamycin resistant degP deletion mutation; and an E. coli strainhaving mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783issued Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g.,PCR or other nucleic acid polymerase reactions, are suitable.

[0148] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forCHEPO-encoding or CHEPO-Ig-encoding vectors. Saccharomyces cerevisiae isa commonly used lower eukaryotic host microorganism. Others includeSchizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 [1981]; EP139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al., Bio/Technology, 9: 968-975 (1991)) such as,e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J.Bacteriol., 737 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8: 135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28: 265-278 [1988]); Candida; Trichoderma reesia (EP 244,234);Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occidentalis(EP 394,538 published Oct. 31, 1990); and filamentous fungi such as,e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan.10 1991), and Aspergillus hosts such as A. nidulans (Ballance et al.,Biochem. Biophys. Res. Commun., 112: 284-289 [1983]; Tilburn et al.,Gene, 26: 205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

[0149] Suitable host cells for the expression of glycosylated CHEPO orCHEPO-Ig are derived from multicellular organisms. Examples ofinvertebrate cells include insect cells such as Drosophila S2 andSpodoptera Sf9, as well as plant cells. Examples of useful mammalianhost cell lines include Chinese hamster ovary (CHO) and COS cells. Morespecific examples include monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. GenVirol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlauband Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertolicells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mousemammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriatehost cell is deemed to be within the skill in the art.

[0150] 4. Selection and Use of a Replicable Vector

[0151] The nucleic acid (e.g., cDNA or genomic DNA) encoding CHEPO or aCHEPO-immunoadhesin may be inserted into a replicable vector for cloning(amplification of the DNA) or for expression. Various vectors arepublicly available. The vector may, for example, be in the form of aplasmid, cosmid, viral particle, or phage. The appropriate nucleic acidsequence may be inserted into the vector by a variety of procedures. Ingeneral, DNA is inserted into an appropriate restriction endonucleasesite(s) using techniques known in the art. Vector components generallyinclude, but are not limited to, one or more of a signal sequence, anorigin of replication, one or more marker genes, an enhancer element, apromoter, and a transcription termination sequence. Construction ofsuitable vectors containing one or more of these components employsstandard ligation techniques which are known to the skilled artisan.

[0152] CHEPO may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe immunoadhesin-encoding DNA that is inserted into the vector. Thesignal sequence may be a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

[0153] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pBR322 issuitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma, adenovirusor BPV) are useful for cloning vectors in mammalian cells.

[0154] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0155] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upCHEPO- or CHEPO-immunoadhesin-encoding nucleic acid, such as DHFR orthymidine kinase. An appropriate host cell when wild-type DHFR isemployed is the CHO cell line deficient in DHFR activity, prepared andpropagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA,77:4216 (1980). A suitable selection gene for use in yeast is the trp1gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature,282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al.,Gene, 10:157 (1980)]. The trp1 gene provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

[0156] Expression and cloning vectors usually contain a promoteroperably linked to CHEPO- or CHEPO-immunoadhesin-encoding nucleic acidsequence to direct mRNA synthesis. Promoters recognized by a variety ofpotential host cells are well known. Promoters suitable for use withprokaryotic hosts include the β-lactamase and lactose promoter systems[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544(1979)], alkaline phosphatase, a tryptophan (trp) promoter system[Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybridpromoters such as the tac promoter [deBoer et al., Proc. Natl. Acad.Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding immunoadhesin.

[0157] Examples of suitable promoter sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0158] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0159] The transcription of CHEPO or CHEPO-immunoadhesin from vectors inmammalian host cells is controlled, for example, by promoters obtainedfrom the genomes of viruses such as polyoma virus, fowlpox virus (UK2,211,504 published Jul. 15, 1989), adenovirus (such as Adenovirus 2),bovine papilloma virus, retrovirus (such as avian sarcoma virus),cytomegalovirus, hepatitis-B virus and Simian Virus 40 (SV40); fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, or from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

[0160] Transcription of a DNA encoding CHEPO or CHEPO-immunoadhesin byhigher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp, that act on a promoter to increase its transcription.Many enhancer sequences are now known from mammalian genes (globin,elastase, albumin, α-fetoprotein, and insulin). Typically, however, onewill use an enhancer from a eukaryotic cell virus. Examples include theSV40 enhancer on the late side of the replication origin (by 100-270),the cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to CHEPOor CHEPO-immunoadhesin coding sequence, but is preferably located at asite 5′ from the promoter.

[0161] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding immunoadhesin.

[0162] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of CHEPO or immunoadhesin in recombinantvertebrate cell culture are described in Gething et al., Nature,293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060;and EP 117,058.

[0163] 5. Detecting Gene Amplification/Expression

[0164] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.,Acad. Sci. USA, 77:5201-52-5 (1980)], dot blotting (DNA analysis), or insitu hybridiation, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0165] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical stainining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequenceCHEPO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to CHEPODNA and encoding a specific antibody epitope.

[0166] 6. Purification of the CHEPO polypeptide

[0167] Forms of CHEPO may be recovered from culture medium or from hostcell lysates. If membrane-bound, it can be released from the membraneusing a suitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of CHEPO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

[0168] It may be desired to purify CHEPO from recombinant cell proteinsor polypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of theCHEPO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, N.Y. (1982). The purificationstep(s) selected will depend, for example, on the nature of theproduction process used and the particular CHEPO produced.

[0169] 7. Purification of CHEPO-immunoadhesins

[0170] An immunoadhesin is typically recovered from the culture mediumas a secreted polypeptide, although it also may be recovered from hostcell lysates. As a first step, the particulate debris, either host cellsor lysed fragments, is removed, for example, by centrifugation orultrafiltration; optionally, the protein may be concentrated with acommercially available protein concentration filter, followed byseparating the immunoadhesin from other impurities by one or morepurification procedures selected from: fractionation on animmunoaffinity column; fractionation on an ion-exchange column; ammoniumsulphate or ethanol precipitation; reverse phase HPLC; chromatography onsilica; chromatography on heparin Sepharose; chromatography on a cationexchange resin; chromatofocusing; SDS-PAGE; and gel filtration.

[0171] A particularly advantageous method of purifying immunoadhesins isaffinity chromatography. The choice of affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human IgG1, IgG2, or IgG4 heavy chains [Lindmark et al., J. Immunol.Meth. 62, 1-13 (1983)]. Protein G is recommended for all mouse isotypesand for human IgG3 [Guss et al., EMBO J. 5, 15671575 (1986)]. The matrixto which the affinity ligand is attached is most often agarose, butother matrices are also available. Mechanically stable matrices such ascontrolled pore glass or poly(styrenedivinyl)benzene allow for fasterflow rates and shorter processing times than can be achieved withagarose. The conditions for binding an immunoadhesin to the protein A orG affinity column are dictated entirely by the characteristics of the Fcdomain; that is, its species and isotype. Generally, when the properligand is chosen, efficient binding occurs directly from unconditionedculture fluid. One distinguishing feature of immunoadhesins is that, forhuman IgG1 molecules, the binding capacity for protein A is somewhatdiminished relative to an antibody of the same Fc type. Boundimmunoadhesin can be efficiently eluted either at acidic pH (at or above3.0), or in a neutral pH buffer containing a mildly chaotropic salt.This affinity chromatography step can result in an immunoadhesinpreparation that is >95% pure.

[0172] Other methods known in the art can be used in place of, or inaddition to, affinity chromatography on protein A or G to purifyimmunoadhesins. Immunoadhesins behave similarly to antibodies inthiophilic gel chromatography [Hutchens and Porath, Anal. Biochem. 159,217-226 (1986)] and immobilized metal chelate chromatography[Al-Mashikhi and Makai, J. Dairy Sci. 71, 1756-1763 (1988)]. In contrastto antibodies, however, their behavior on ion exchange columns isdictated not only by their isoelectric points, but also by a chargedipole that may exist in the molecules due to their chimeric nature.

[0173] Preparation of epitope tagged immunoadhesin, such as CHEPO-IgG,facilitates purification using an immunoaffinity column containingantibody to the epitope to adsorb the fusion polypeptide. Immunoaffinitycolumns such as a rabbit polyclonal anti-CHEPO column can be employed toabsorb the CHEPO-IgG by binding it to an CHEPO immune epitope.

[0174] E. Uses for CHEPO

[0175] Nucleotide sequences (or their complement) encoding CHEPO havevarious applications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. CHEPO nucleic acid will also beuseful for the preparation of CHEPO polypeptides by the recombinanttechniques described herein.

[0176] The full-length native sequence CHEPO cDNA (SEQ ID NO:3), orportions thereof, may be used as hybridization probes for a cDNA libraryto isolate the full-length CHEPO cDNA or to isolate still other cDNAs(for instance, those encoding naturally-occurring variants of CHEPO orCHEPO from other species) which have a desired sequence identity to theCHEPO sequence disclosed in FIG. 2 (SEQ ID NO:3). Optionally, the lengthof the probes will be about 20 to about 50 bases. The hybridizationprobes may be derived from at least partially novel regions of thenucleotide sequence of SEQ ID NO:3 wherein those regions may bedetermined without undue experimentation or from genomic sequencesincluding promoters, enhancer elements and introns of native sequenceCHEPO. By way of example, a screening method will comprise isolating thecoding region of the CHEPO gene using the known DNA sequence tosynthesize a selected probe of about 40 bases. Hybridization probes maybe labeled by a variety of labels, including radionucleotides such as³²P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems. Labeled probes having asequence complementary to that of the CHEPO gene of the presentinvention can be used to screen libraries of human cDNA, genomic DNA ormRNA to determine which members of such libraries the probe hybridizesto. Hybridization techniques are described in further detail in theExamples below.

[0177] Any EST sequences disclosed in the present application maysimilarly be employed as probes, using the methods disclosed herein.

[0178] Other useful fragments of the CHEPO nucleic acids includeantisense or sense oligonucleotides comprising a singe-stranded nucleicacid sequence (either RNA or DNA) capable of binding to target CHEPOmRNA (sense) or CHEPO DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of CHEPO DNA. Such a fragment generallycomprises at least about 14 nucleotides, preferably from about 14 to 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0179] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranscription or translation of the target sequence by one of severalmeans, including enhanced degradation of the duplexes, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression of CHEPOproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0180] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10048, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0181] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

[0182] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0183] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0184] The probes may also be employed in PCR techniques to generate apool of sequences for identification of closely related CHEPO codingsequences.

[0185] Nucleotide sequences encoding a CHEPO can also be used toconstruct hybridization probes for mapping the gene which encodes thatCHEPO and for the genetic analysis of individuals with geneticdisorders. The nucleotide sequences provided herein may be mapped to achromosome and specific regions of a chromosome using known techniques,such as in situ hybridization, linkage analysis against knownchromosomal markers, and hybridization screening with libraries.

[0186] When the coding sequences for CHEPO encode a protein which bindsto another protein (example, where the CHEPO is a receptor), the CHEPOcan be used in assays to identify the other proteins or moleculesinvolved in the binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor CHEPO can be used to isolate correlative ligand(s).Screening assays can be designed to find lead compounds that mimic thebiological activity of a native CHEPO or a receptor for CHEPO. Suchscreening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates. Small molecules contemplatedinclude synthetic organic or inorganic compounds. The assays can beperformed in a variety of formats, including protein-protein bindingassays, biochemical screening assays, immunoassays and cell basedassays, which are well characterized in the art.

[0187] Nucleic acids which encode CHEPO or its modified forms can alsobe used to generate either transgenic animals or “knock out” animalswhich, in turn, are useful in the development and screening oftherapeutically useful reagents. A transgenic animal (e.g., a mouse orrat) is an animal having cells that contain a transgene, which transgenewas introduced into the animal or an ancestor of the animal at aprenatal, e.g., an embryonic stage. A transgene is a DNA which isintegrated into the genome of a cell from which a transgenic animaldevelops. In one embodiment, cDNA encoding CHEPO can be used to clonegenomic DNA encoding CHEPO in accordance with established techniques andthe genomic sequences used to generate transgenic animals that containcells which express DNA encoding CHEPO. Methods for generatingtransgenic animals, particularly animals such as mice or rats, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells would betargeted for CHEPO transgene incorporation with tissue-specificenhancers. Transgenic animals that include a copy of a transgeneencoding CHEPO introduced into the germ line of the animal at anembryonic stage can be used to examine the effect of increasedexpression of DNA encoding CHEPO. Such animals can be used as testeranimals for reagents thought to confer protection from, for example,pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

[0188] Alternatively, non-human homologues of CHEPO can be used toconstruct a CHEPO “knock out” animal which has a defective or alteredgene encoding CHEPO as a result of homologous recombination between theendogenous gene encoding CHEPO and altered genomic DNA encoding CHEPOintroduced into an embryonic stem cell of the animal. For example, cDNAencoding CHEPO can be used to clone genomic DNA encoding CHEPO inaccordance with established techniques. A portion of the genomic DNAencoding CHEPO can be deleted or replaced with another gene, such as agene encoding a selectable marker which can be used to monitorintegration. Typically, several kilobases of unaltered flanking DNA(both at the 5′ and 3′ ends) are included in the vector [see e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors]. The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected[see e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras [see e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152]. A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the CHEPO polypeptide.

[0189] Nucleic acid encoding the CHEPO polypeptides may also be used ingene therapy. In gene therapy applications, genes are introduced intocells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

[0190] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology 11, 205-210 [1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0191] The CHEPO polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes.

[0192] The nucleic acid molecules encoding the CHEPO polypeptides orfragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identifynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachCHEPO nucleic acid molecule of the present invention can be used as achromosome marker.

[0193] The CHEPO polypeptides and nucleic acid molecules of the presentinvention may also be used for tissue typing, wherein the CHEPOpolypeptides of the present invention may be differentially expressed inone tissue as compared to another. CHEPO nucleic acid molecules willfind use for generating probes for PCR, Northern analysis, Southernanalysis and Western analysis.

[0194] The CHEPO polypeptides described herein may also be employed astherapeutic agents. The CHEPO polypeptides of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the CHEPO product hereof is combined inadmixture with a pharmaceutically acceptable carrier vehicle.Therapeutic formulations are prepared for storage by mixing the activeingredient having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™ or PEG.

[0195] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

[0196] Therapeutic compositions herein generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0197] The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

[0198] Dosages and desired drug concentrations of pharmaceuticalcompositions of the present invention may vary depending on theparticular use envisioned. The determination of the appropriate dosageor route of administration is well within the skill of an ordinaryphysician. Animal experiments provide reliable guidance for thedetermination of effective doses for human therapy. Interspecies scalingof effective doses can be performed following the principles laid downby Mordenti, J. and Chappell, W. “The use of interspecies scaling intoxicokinetics” In Toxicokinetics and New Drug Development, Yacobi etal., Eds., Pergamon Press, New York 1989, pp. 42-96.

[0199] When in vivo administration of a CHEPO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

[0200] Where sustained-release administration of a CHEPO polypeptide isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of theCHEPO polypeptide, microencapsulation of the CHEPO polypeptide iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon- (rhIFN- ), interleukin-2, and MN rgp120. Johnson etal., Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. Ther., 27: 1221-1223(1993); Hora et al., Bio/Technology. 8: 755-758 (1990); Cleland, “Designand Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S Pat.No. 5,654,010.

[0201] The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

[0202] This invention encompasses methods of screening compounds toidentify those that mimic the CHEPO polypeptide (agonists) or preventthe effect of the CHEPO polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the CHEPO polypeptides encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

[0203] The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

[0204] All assays for antagonists are common in that they call forcontacting the drug candidate with a CHEPO polypeptide encoded by anucleic acid identified herein under conditions and for a timesufficient to allow these two components to interact.

[0205] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the CHEPO polypeptide encoded by the geneidentified herein or the drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.Non-covalent attachment generally is accomplished by coating the solidsurface with a solution of the CHEPO polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the CHEPO polypeptide to be immobilized can be used toanchor it to a solid surface. The assay is performed by adding thenon-immobilized component, which may be labeled by a detectable label,to the immobilized component, e.g., the coated surface containing theanchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

[0206] If the candidate compound interacts with but does not bind to aparticular CHEPO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340: 245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

[0207] Compounds that interfere with the interaction of a gene encodinga CHEPO polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

[0208] To assay for antagonists, the CHEPO polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the CHEPO polypeptide indicates that the compound is anantagonist to the CHEPO polypeptide. Alternatively, antagonists may bedetected by combining the CHEPO polypeptide and a potential antagonistwith membrane-bound CHEPO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. TheCHEPO polypeptide can be labeled, such as by radioactivity, such thatthe number of CHEPO polypeptide molecules bound to the receptor can beused to determine the effectiveness of the potential antagonist. Thegene encoding the receptor can be identified by numerous methods knownto those of skill in the art, for example, ligand panning and FACSsorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5(1991). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to the CHEPOpolypeptide and a cDNA library created from this RNA is divided intopools and used to transfect COS cells or other cells that are notresponsive to the CHEPO polypeptide. Transfected cells that are grown onglass slides are exposed to labeled CHEPO polypeptide. The CHEPOpolypeptide can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an interactive sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor.

[0209] As an alternative approach for receptor identification, labeledCHEPO polypeptide can be photoaffinty-linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE and exposed to X-ray film. The labeledcomplex containing the receptor can be excised, resolved into peptidefragments, and subjected to protein micro-sequencing. The amino acidsequence obtained from micro- sequencing would be used to design a setof degenerate oligonucleotide probes to screen a cDNA library toidentify the gene encoding the putative receptor.

[0210] In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeledCHEPO polypeptide in the presence of the candidate compound. The abilityof the compound to enhance or block this interaction could then bemeasured.

[0211] More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with CHEPOpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of theCHEPO polypeptide that recognizes the receptor but imparts no effect,thereby competitively inhibiting the action of the CHEPO polypeptide.

[0212] Another potential CHEPO polypeptide antagonist is an antisenseRNA or DNA construct prepared using antisense technology, where, e.g.,an antisense RNA or DNA molecule acts to block directly the translationof mRNA by hybridizing to targeted mRNA and preventing proteintranslation. Antisense technology can be used to control gene expressionthrough triple-helix formation or antisense DNA or RNA, both of whichmethods are based on binding of a polynucleotide to DNA or RNA. Forexample, the 5′ coding portion of the polynucleotide sequence, whichencodes the mature CHEPO polypeptides herein, is used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription (triple helix—see Lee etal., Nucl. Acids Res., 6: 3073 (1979); Cooney et al., Science, 241: 456(1988); Dervan et al., Science, 251: 1360 (1991)), thereby preventingtranscription and the production of the CHEPO polypeptide. The antisenseRNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the CHEPO polypeptide(antisense—Okano, Neurochem., 56: 560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla.,1988). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of the CHEPO polypeptide. When antisense DNA is used,oligodeoxyribonucleotides derived from the translation-initiation site,e.g., between about −10 and +10 positions of the target gene nucleotidesequence, are preferred.

[0213] Potential antagonists include small molecules that bind to theactive site, the receptor binding site, or growth factor or otherrelevant binding site of the CHEPO polypeptide, thereby blocking thenormal biological activity of the CHEPO polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

[0214] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g., Rossi, Current Biology, 4: 469-471 (1994), and PCTpublication No. WO 97/33551 (published Sep.18, 1997).

[0215] Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

[0216] These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0217] F. Anti-CHEPO Antibodies

[0218] The present invention further provides anti-CHEPO antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

[0219] 1. Polyclonal Antibodies

[0220] The anti-CHEPO antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the CHEPO polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0221] 2. Monoclonal Antibodies

[0222] The anti-CHEPO antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0223] The immunizing agent will typically include the CHEPO polypeptideor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

[0224] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0225] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst CHEPO. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0226] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0227] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0228] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0229] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0230] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0231] 3. Human and Humanized Antibodies

[0232] The anti-CHEPO antibodies of the invention may further comprisehumanized antibodies or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0233] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeye et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0234] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boemer et al. arealso available for the preparation of human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-783(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0235] 4. Bispecific Antibodies

[0236] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the CHEPO, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

[0237] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0238] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0239] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain(s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0240] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared can be prepared using chemical linkage. Brennan et al., Science229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0241] Fab′ fragments may be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217-225 (1992) describe the production of a fullyhumanized bispecific antibody F(ab′)₂ molecule. Each Fab′ fragment wasseparately secreted from E. coli and subjected to directed chemicalcoupling in vitro to form the bispecific antibody. The bispecificantibody thus formed was able to bind to cells overexpressing the ErbB2receptor and normal human T cells, as well as trigger the lytic activityof human cytotoxic lymphocytes against human breast tumor targets.

[0242] Various technique for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodieswith more than two valencies are contemplated. For example, trispecificantibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0243] Exemplary bispecific antibodies may bind to two differentepitopes on a given CHEPO polypeptide herein. Alternatively, ananti-CHEPO polypeptide arm may be combined with an arm which binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcγR), such asFcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellulardefense mechanisms to the cell expressing the particular CHEPOpolypeptide. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express a particular CHEPO polypeptide.These antibodies possess a CHEPO-binding arm and an arm which binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA,or TETA. Another bispecific antibody of interest binds the CHEPOpolypeptide and further binds tissue factor (TF).

[0244] 5. Heteroconiugate Antibodies

[0245] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0246] 6. Effector Function Engineering

[0247] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

[0248] 7. Immunoconjugates

[0249] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0250] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re.

[0251] Conjugates of the antibody and cytotoxic agent are made using avariety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinmidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamnine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

[0252] In another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0253] 8. Immunoliposomes

[0254] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0255] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

[0256] 9. Pharmaceutical Compositions of Antibodies

[0257] Antibodies specifically binding a CHEPO polypeptide identifiedherein, as well as other molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders in the form of pharmaceutical compositions.

[0258] If the CHEPO polypeptide is intracellular and whole antibodiesare used as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Nati. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0259] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0260] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0261] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0262] G. Uses for Anti-CHEPO Antibodies

[0263] The anti-CHEPO antibodies of the invention have variousutilities. For example, anti-CHEPO antibodies may be used in diagnosticassays for CHEPO, e.g., detecting its expression in specific cells,tissues, or serum. Various diagnostic assay techniques known in the artmay be used, such as competitive binding assays, direct or indirectsandwich assays and immunoprecipitation assays conducted in eitherheterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: AManual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietymay be employed, including those methods described by Hunter et al.,Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Painet al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0264] Anti-CHEPO antibodies also are useful for the affinitypurification of CHEPO from recombinant cell culture or natural sources.In this process, the antibodies against CHEPO are immobilized on asuitable support, such a Sephadex resin or filter paper, using methodswell known in the art. The immobilized antibody then is contacted with asample containing the CHEPO to be purified, and thereafter the supportis washed with a suitable solvent that will remove substantially all thematerial in the sample except the CHEPO, which is bound to theimmobilized antibody. Finally, the support is washed with anothersuitable solvent that will release the CHEPO from the antibody.

[0265] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0266] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0267] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, Va.

Example 1 Isolation of Nucleic Acid Encoding CHEPO

[0268] Genomic DNA was isolated from two chimpanzee cell lines (ATCCCRL1609 and CRL1857) using a Qiagen kit (cat#10262) as recommended bythe manufacturer's instructions. The chimp Epo gene was then obtained on3 separate fragments by PCR using 1 μg of genomic DNA as template andthe following primer pairs: EPO.F: 5′-ACCGCGCCCCCTGGACAG-3′ (SEQ IDNO:12) EPO.INT1.R: 5′-CATCCACTTCTCCGGCCAAACTTCA-3′ (SEQ ID NO:13)EPO.INT1F: 5′-TTTGGCCGGAGAAGTGGATGC-3′ (SEQ ID NO:14) EPO.INT4R:5′-TCACTCACTCACTCATTCATTCATTCATTCA-3′ (SEQ ID NO:15) EPO.INT4F:5′-GTTGAATGAATGATTGAATGAATGAGTGA-3′ (SEQ ID NO:16) EPO.R:5′-GCACTGGAGTGTCCATGGGACAG-3′ (SEQ ID NO:17)

[0269] Each PCR reaction contained 5 μl of 10× PCR Buffer (PerkinElmer), 1 μl dNTP (2 mM), 1 μg genomic DNA, 1 μl of each primer, 1 μl ofTaq polymerase (Clontech) and H₂O to bring the total volume to 50μl. Thereaction was first denatured for 4 min. at 94° C. then amplified for 40cycles of 1 min. at 94° C., 1 min. at 65° C. or 66° C. then extended for1 min. at 72° C. A last extension step of 5 min. at 72° C. wasperformed. The reaction was then analyzed on agarose gel. PCR product of500 bp, 1200 bp and 750 bp were observed for each PCR product,respectively. The PCR reactions were then purified using a Wizard kit(Promega cat # A7170) then directly sequenced. DNA sequencing of the PCRproducts was done using an Applied Biosystems 377 DNA Sequencer(PE/Applied Biosystems, Foster City, Calif.). The chemistry used was DYETerminator Cycle Sequencing with dRhodamine and BIG DYE terminators(PE/Applied Biosystems, Foster City, Calif.). Sequence assembly andediting done with Sequencher software (Gene Codes, Ann Arbor, Mich.).

[0270] The 5 coding exons were identified by homology with the humanerythropoietin sequence and assembled into a predicted full length cDNA.The coding region of CHEPO cDNA is 579 nucleotides long (FIG. 1) andencodes a predicted protein of 193 amino acids (FIG. 3). There are 3putative signal cleavage sites predicted after amino acid residue 22, 24and 27. In accordance with the N-terminus of human Epo, the latest oneis likely to correspond to the cleavage site for chimp Epo. Thehydrophobic 27 amino acid signal peptide is followed by a 166 amino acidlong mature protein containing 3 potential N-glycosylation sites. Asingle nucleotide polymorphism is present in the predicted sequenceobtained from CRL1609 and changes the protein sequence at amino acidposition 142 from a Q to a K. Alignment of the chimp Epo protein withthe human sequence indicates a single change at amino acid position 84(FIG. 3) between the 2 sequences.

Example 2 Use of CHEPO cDNA as a Hybridization Probe

[0271] The following method describes use of a nucleotide sequenceencoding CHEPO as a hybridization probe.

[0272] DNA comprising the coding sequence of full-length or mature CHEPO(as shown in FIG. 2, SEQ ID NO:3) is employed as a probe to screen forhomologous DNAs (such as those encoding naturally-occurring variants ofCHEPO) in human tissue cDNA libraries or human tissue genomic libraries.

[0273] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled CHEPO-derived probe to the filters isperformed in a solution of 50% formamide, 5×SSC, 0.1 % SDS, 0.1% sodiumpyrophosphate, 50 mM sodium phosphate, pH 6.8, 2×Denhardt's solution,and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filtersis performed in an aqueous solution of 0.1×SSC and 0.1% SDS at 42° C.

[0274] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence CHEPO can then be identified using standardtechniques known in the art.

Example 3 Expression of CHEPO in E. coli

[0275] This example illustrates preparation of an unglycosylated form ofCHEPO by recombinant expression in E. coli.

[0276] The DNA sequence encoding CHEPO (SEQ ID NO:3) is initiallyamplified using selected PCR primers. The primers should containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector. A variety of expression vectorsmay be employed. An example of a suitable vector is pBR322 (derived fromE. coli ; see Bolivar et al., Gene, 2:95 (1977)) which contains genesfor ampicillin and tetracycline resistance. The vector is digested withrestriction enzyme and dephosphorylated. The PCR amplified sequences arethen ligated into the vector. The vector will preferably includesequences which encode for an antibiotic resistance gene, a trppromoter, a polyhis leader (including the first six STII codons, polyhissequence, and enterokinase cleavage site), the CHEPO coding region,lambda transcriptional terminator, and an argU gene.

[0277] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0278] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0279] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized CHEPO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

[0280] CHEPO may be expressed in E. coli in a poly-His tagged form,using the following procedure. The DNA encoding CHEPO is initiallyamplified using selected PCR primers. The primers will containrestriction enzyme sites which correspond to the restriction enzymesites on the selected expression vector, and other useful sequencesproviding for efficient and reliable translation initiation, rapidpurification on a metal chelation column, and proteolytic removal withenterokinase. The PCR-amplified, poly-His tagged sequences are thenligated into an expression vector, which is used to transform an E. colihost based on strain 52 (W3110 fuhA(tonA) lon galE rpoHts(htpRts)clpP(lacIq). Transformants are first grown in LB containing 50 mg/mlcarbenicillin at 30° C. with shaking until an O.D.600 of 3-5 is reached.Cultures are then diluted 50-100 fold into CRAP media (prepared bymixing 3.57 g (NH₄)₂SO₄, 0.71 g sodium citrate.2H2O, 1.07 g KCl, 5.36 gDifco yeast extract, 5.36 g Sheffield hycase SF in 500 mL water, as wellas 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grownfor approximately 20-30 hours at 30° C. with shaking. Samples areremoved to verify expression by SDS-PAGE analysis, and the bulk cultureis centrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

[0281]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1 M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

[0282] The proteins are refolded by diluting the sample slowly intofreshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA.Refolding volumes are chosen so that the final protein concentration isbetween 50 to 100 micrograms/mil. The refolding solution is stirredgently at 4° C. for 12-36 hours. The refolding reaction is quenched bythe addition of TFA to a final concentration of 0.4% (pH ofapproximately 3). Before further purification of the protein, thesolution is filtered through a 0.22 micron filter and acetonitrile isadded to 2-10% final concentration. The refolded protein ischromatographed on a Poros R1/H reversed phase column using a mobilebuffer of 0.1% TFA with elution with a gradient of acetonitrile from 10to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDSpolyacrylamide gels and fractions containing homogeneous refoldedprotein are pooled. Generally, the properly refolded species of mostproteins are eluted at the lowest concentrations of acetonitrile sincethose species are the most compact with their hydrophobic interiorsshielded from interaction with the reversed phase resin. Aggregatedspecies are usually eluted at higher acetonitrile concentrations. Inaddition to resolving misfolded forms of proteins from the desired form,the reversed phase step also removes endotoxin from the samples.

[0283] Fractions containing the desired folded CHEPO polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

Example 4 Expression of CHEPO in Mammalian Cells

[0284] This example illustrates preparation of a potentiallyglycosylated form of CHEPO by recombinant expression in mammalian cells.

[0285] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the CHEPO DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of theCHEPO DNA using ligation methods such as described in Sambrook et al.,supra. The resulting vector is called pRK5-CHEPO.

[0286] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-CHEPO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO₄,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

[0287] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of CHEPO polypeptide. The culturescontaining transfected cells may undergo further incubation (in serumfree medium) and the medium is tested in selected bioassays.

[0288] In an alternative technique, CHEPO may be introduced into 293cells transiently using the dextran sulfate method described bySomparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells aregrown to maximal density in a spinner flask and 700 μg pRK5-CHEPO DNA isadded. The cells are first concentrated from the spinner flask bycentrifugation and washed with PBS. The DNA-dextran precipitate isincubated on the cell pellet for four hours. The cells are treated with20% glycerol for 90 seconds, washed with tissue culture medium, andre-introduced into the spinner flask containing tissue culture medium, 5μg/ml bovine insulin and 0.1 μg/ml bovine transferrin. After about fourdays, the conditioned media is centrifuged and filtered to remove cellsand debris. The sample containing expressed CHEPO can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

[0289] In another embodiment, CHEPO can be expressed in CHO cells. ThepRK5-CHEPO can be transfected into CHO cells using known reagents suchas CaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of CHEPO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed CHEPO can then be concentrated and purified byany selected method.

[0290] Epitope-tagged CHEPO may also be expressed in host CHO cells. TheCHEPO may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedCHEPO insert can then be subcloned into a SV40 driven vector containinga selection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedCHEPO can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

[0291] CHEPO may also be expressed in CHO and/or COS cells by atransient expression procedure or in CHO cells by another stableexpression procedure.

[0292] Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

[0293] Following PCR amplification, the respective DNAs are subcloned ina CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allowthe convenient shuttling of cDNA's. The vector used expression in CHOcells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

[0294] Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfece® (Quiagen), Dosper® or Fugen®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

[0295] The ampules containing the plasmid DNA are thawed by placementinto water bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 urm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Coming 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 ,μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

[0296] For the poly-His tagged constructs, the proteins are purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole is addedto the conditioned media to a concentration of 5 mM. The conditionedmedia is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column is washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0297] Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 5 Expression of CHEPO in Yeast

[0298] The following method describes recombinant expression of CHEPO inyeast.

[0299] First, yeast expression vectors are constructed for intracellularproduction or secretion of CHEPO from the ADH2/GAPDH promoter. DNAencoding CHEPO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof CHEPO. For secretion, DNA encoding CHEPO can be cloned into theselected plasmid, together with DNA encoding the ADH2/GAPDH promoter, anative CHEPO signal peptide or other mammalian signal peptide, or, forexample, a yeast alpha-factor or invertase secretory signal/leadersequence, and linker sequences (if needed) for expression of CHEPO.

[0300] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0301] Recombinant CHEPO can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing CHEPO may further be purified using selectedcolumn chromatography resins.

Example 6 Expression of CHEPO in Baculovirus-Infected Insect Cells

[0302] The following method describes recombinant expression of CHEPO inBaculovirus-infected insect cells.

[0303] The sequence coding for CHEPO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding CHEPO or the desired portion of the coding sequence ofCHEPO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

[0304] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold T virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0305] Expressed poly-his tagged CHEPO can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows. Extractsare prepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged CHEPO are pooled and dialyzed againstloading buffer.

[0306] Alternatively, purification of the IgG tagged (or Fc tagged)CHEPO can be performed using known chromatography techniques, includingfor instance, Protein A or protein G column chromatography.

Example 7 Preparation of Antibodies that Bind CHEPO

[0307] This example illustrates preparation of monoclonal antibodieswhich can specifically bind CHEPO.

[0308] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified CHEPO, fusion proteins containingCHEPO, and cells expressing recombinant CHEPO on the cell surface.Selection of the immunogen can be made by the skilled artisan withoutundue experimentation.

[0309] Mice, such as Balb/c, are immunized with the CHEPO immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-CHEPO antibodies.

[0310] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of CHEPO. Three to four days later, the mice are sacrificedand the spleen cells are harvested. The spleen cells are then fused(using 35% polyethylene glycol) to a selected murine myeloma cell linesuch as P3X63AgU.1 , available from ATCC, No. CRL 1597. The fusionsgenerate hybridoma cells which can then be plated in 96 well tissueculture plates containing HAT (hypoxanthine, aminopterin, and thymidine)medium to inhibit proliferation of non-fused cells, myeloma hybrids, andspleen cell hybrids.

[0311] The hybridoma cells will be screened in an ELISA for reactivityagainst CHEPO. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against CHEPO is within the skill in theart.

[0312] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing the anti-CHEPOmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 8 Purification of CHEPO Polypeptides Using Specific Antibodies

[0313] Native or recombinant CHEPO polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-CHEPO polypeptide, mature CHEPO polypeptide, or pre-CHEPOpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the CHEPO polypeptide of interest. In general,an immunoaffinity column is constructed by covalently coupling theanti-CHEPO polypeptide antibody to an activated chromatographic resin.

[0314] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0315] Such an immunoaffinity column is utilized in the purification ofCHEPO polypeptide by preparing a fraction from cells containing CHEPOpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble CHEPO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0316] A soluble CHEPO polypeptide-containing preparation is passed overthe immunoaffinity column, and the column is washed under conditionsthat allow the preferential absorbance of CHEPO polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/CHEPO polypeptidebinding (e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and CHEPOpolypeptide is collected.

Example 9 Drug Screening

[0317] This invention is particularly useful for screening compounds byusing CHEPO polypeptides or binding fragment thereof in any of a varietyof drug screening techniques. The CHEPO polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing theCHEPO polypeptide or fragment. Drugs are screened against suchtransformed cells in competitive binding assays. Such cells, either inviable or fixed form, can be used for standard binding assays. One maymeasure, for example, the formation of complexes between CHEPOpolypeptide or a fragment and the agent being tested. Alternatively, onecan examine the diminution in complex formation between the CHEPOpolypeptide and its target cell or target receptors caused by the agentbeing tested.

[0318] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect a CHEPOpolypeptide-associated disease or disorder. These methods comprisecontacting such an agent with an CHEPO polypeptide or fragment thereofand assaying (I) for the presence of a complex between the agent and theCHEPO polypeptide or fragment, or (ii) for the presence of a complexbetween the CHEPO polypeptide or fragment and the cell, by methods wellknown in the art. In such competitive binding assays, the CHEPOpolypeptide or fragment is typically labeled. After suitable incubation,free CHEPO polypeptide or fragment is separated from that present inbound form, and the amount of free or uncomplexed label is a measure ofthe ability of the particular agent to bind to CHEPO polypeptide or tointerfere with the CHEPO polypeptide/cell complex.

[0319] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to apolypeptide and is described in detail in WO 84/03564, published onSept. 13, 1984. Briefly stated, large numbers of different small peptidetest compounds are synthesized on a solid substrate, such as plasticpins or some other surface. As applied to a CHEPO polypeptide, thepeptide test compounds are reacted with CHEPO polypeptide and washed.Bound CHEPO polypeptide is detected by methods well known in the art.Purified CHEPO polypeptide can also be coated directly onto plates foruse in the aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

[0320] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of bindingCHEPO polypeptide specifically compete with a test compound for bindingto CHEPO polypeptide or fragments thereof. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with CHEPO polypeptide.

Example 10 Rational Drug Design

[0321] The goal of rational drug design is to produce structural analogsof biologically active polypeptide of interest (i.e., a CHEPOpolypeptide) or of small molecules with which they interact, e.g.,agonists, antagonists, or inhibitors. Any of these examples can be usedto fashion drugs which are more active or stable forms of the CHEPOpolypeptide or which enhance or interfere with the function of the CHEPOpolypeptide in vivo (cf., Hodgson, Bio/Technology, 9: 19-21 (1991)).

[0322] In one approach, the three-dimensional structure of the CHEPOpolypeptide, or of an CHEPO polypeptide-inhibitor complex, is determinedby x-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of theCHEPO polypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the CHEPO polypeptide may be gained bymodeling based on the structure of homologous proteins. In both cases,relevant structural information is used to design analogous CHEPOpolypeptide-like molecules or to identify efficient inhibitors. Usefulexamples of rational drug design may include molecules which haveimproved activity or stability as shown by Braxton and Wells,Biochemistry, 31:7796-7801 (1992) or which act as inhibitors, agonists,or antagonists of native peptides as shown by Athauda et al., J.Biochem., 113:742-746 (1993).

[0323] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

[0324] By virtue of the present invention, sufficient amounts of theCHEPO polypeptide may be made available to perform such analyticalstudies as X-ray crystallography. In addition, knowledge of the CHEPOpolypeptide amino acid sequence provided herein will provide guidance tothose employing computer modeling techniques in place of or in additionto x-ray crystallography.

[0325] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention asclaimed. Various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

Example 11 Preparation of a CHEPO-Immunoadhesin

[0326] In order to construct a CHEPO polypeptide having an enhanced invivo half-life, a CHEPO immunoadhesin molecule was constructed asfollows.

[0327] A. Assembly of CHEPO cDNA

[0328] The chimpanzee Epo (CHEPO) gene was cloned as described inExample 1.

[0329] An expression construct containing the CHEPO gene under thecontrol of the CMV promoter was generated by amplifying the CHEPO genefrom chimp genomic DNA with the following forward and reverse primers:Eco-CHEPO.F: 5′-CGGAATTCATGGGGGTGCACGAATGTCCTGCCTGGC-3′ (SEQ ID NO: 50)and Xba-CHEPO.R: 5′-GCTCTAGACTCATCTGTCCCCTGTCCTGCAGG-3′ (SEQ ID NO: 51).

[0330] The forward primer corresponds to the coding region of Exon 1fused in frame to the beginning of Exon 2. The reverse primercorresponds to the carboxy terminus of Exon 5, including the stop codon.

[0331] The ⁻1.6 kb PCR product therefore corresponds to the CHEPO gene,beginning with the the ATG of Exon 1 and ending with the Stop codon ofExon 5, and without Intron 1. The PCR product was subcloned into pRK5after digestion with the restriction enzymes, EcoRI and XbaI, andtransformed into bacteria. DNA isolated from 4 independent clones wassubjected to DNA sequencing. DNA from one clone with the correctsequence was selected (clone #6) and transfected by the calciumphosphate method into 293 cells. Total RNA was isolated from a 10 cmplate of transfected cells using RNA STAT-60 (Tel-Test Inc.,Friendswood, Tex.) following instructions from the manufacturer. Afterethanol precipitation, the RNA pellet was resuspended in 250 μlDEPC-treated water and with a Perkin-Elmer GeneAmp RNA PCR core kit wassubjected to reverse transcription, resulting in CHEPO cDNA. The CHEPOcDNA was then amplified using Eco-CHEPO.F and Xba-CHEPO.R as primers.Upon separation of the amplified products by gel electrophoresis,several bands were detected. A DNA band of the predicted size for thecorrectly spliced CHEPO gene (approximately 0.5 kb) was isolated fromthe gel and subcloned into pRK5 after digestion with the restrictionenzymes, EcoRI and XbaI, and tranformed into bacteria. DNA isolated fromclones were subjected to DNA sequencing. A clone, clone 3H3, with thecorrect sequence was selected for further study.

[0332] B. Construction of CHEPO-IgG1

[0333] The following method describes construction of a CHEPO-IgGimmunoadhesin. The coding region of CHEPO was amplified from clone 3H3,described above, with Pfu polymerase using Eco-CHEPO.F as the forwardprimer and the following reverse primer: BstE2.CHEPO.R:5′-GTCCGGGTGACCCCTCTGTCCCCTGTCCTGCAGGC-3′ (SEQ ID NO: 52)

[0334] The BstE2.CHEPO.R primer corresponds to the carboxy terminus ofExon 5 of the CHEPO sequence with the 5′ end of the primer beginningimmediately upstream of the stop codon of Exon 5.

[0335] The PCR product was digested with the restriction enzymes, EcoRIand BstE2, and subcloned into the vector, pBSSK-CH2CH3, in frame withhuman IgG1 -Fc to create pBSSK-CH2CH3-CHEPO-IgG1. DNA isolated frombacterial clones expressing the vector containing the CHEPO-IgG1fusion,pBSSK-CH2CH3-CHEPO-IgG1 was subjected to DNA sequencing. One clone withthe correct sequence (#3) was selected for further study. DNA from clone#3 was digested with the restriction enzymes, EcoRI and XbaI, to releasethe CHEPO-IgG1 product. The digested product encoding CEPO-IgG1 wassubcloned into pRK5, generating pRK5-CHEPO-IgG1. pRK5-CHEPO-IgG1wastransiently transfected in 293 cells by the calcium phosphate method,described above, and CHEPO-IgG1 was purified from the conditioned mediausing methods that involve protein A chromatography.

Example 12 Biological Activity of CHEPO-IgG1

[0336] The biological activity of CHEPO-IgG1was evaluated by threeindependent assays, measuring the ability of CHEPO-IgG1to stimulate 1)the proliferation of Ba/F3-EpoR cells 2) the production of erythroidcolonies from human bone marrow cells, and 3) the formation of immatureand mature erythroid cells in liquid cultures of bone marrow cells.

[0337] A DNA fragment corresponding to the entire coding sequence ofhuman EPO receptor (EpoR) was obtained by PCR and cloned into pRK5-tkneo(a pRK5 vector containing the tymidine kinase (tk)-neo marker) to createa vector designated pRK5-tkneo EpoR. After linearization by digestionwith restriction enzymes, the pRK5-tkneo EpoR construct was introducedinto Ba/F3 cells by electroporation (250 Volts, 960 μF). Neomycinresistant cells, expressing the pRK5-tkneo EpoR construct, were selectedin 2 mg/ml G418, and individual clones were obtained by limitingdilutions.

[0338] A. Stimulation of proliferation of Ba/F3-EpoR cells

[0339] Clones expressing EpoR from the pRK5-tkneo EpoR construct wereanalyzed for their responses to recombinant human Epo (rhEpo) (Amgen) orchimpanzee-IgG1 (CHEPO-IgG1), prepared as described in the previousexample, in a biological assay measuring stimulation of proliferation ofBa/F3-EpoR cells. The cells were developed internally. Briefl, theentire ORF of human EpoR was amplified by PCR and subcloned into apRK5tkneo vector (Holmes, Science, 253: 1278-1280 (1991)). The constructwas linearized with NotI and electroporated into Ba/F3 cells. Clonesresistant to neomycin were obtained by limiting dilutions and weretested for their ability to proliferate in response to rhEpo. The bestresponder was eelected for further studies. Stimulation of proliferationof Ba/F3-EpoR cells in response to rhEpo and CHEPO-IgG1 , respectively,was measured by the extent of incorporation of [³H]-thymidine into thecellular DNA. Cells were initially starved of IL-3 for 16 hours, andsubsequently, seeded in 96 well plates at a density of 25,000 cells/wellin media containing rhEpo or CHEPO-IgG1at various concentrations. Afterincubation for 22 hours, 1 μCi of [³H]-thymidine/well were added to thewells, and the cells were incubated for an additional 6 hours beforebeing harvested. Cell-incorporated radioactivity was determined in thepresence of 40 μl of scintillation fluid (μicroscint 20) using a TopCount Counter (Packard Instruments). The average of duplicate resultsfrom the assay are shown in FIG. 4. The efficiency of CHEPO-IgG1 instimulating incorporation of tritiated thymidine in Ba/F3-EpoR cellswere compared to the efficiency of stimulation by rhEpo (FIG. 4) andindicated that the fusion of CHEPO to the Fc region of an IgG1 moleculedoes not affect binding and activation of the Epo receptor.

[0340] B. Stimulation of erythroid colonies in human bone marrow cells

[0341] In an alternate assay for measuring the biological activity ofthe CHEPO-IgG1 immunoadhesin, stimulation of the production of erythroidcolonies by CHEPO-IgG1was monitored in human bone marrow cells. Freshhuman bone marrow aspirates were obtained from healthy donors (PoieticTechnologies, Gaithersburg, Md.). The mononuclear fraction was enrichedfor CD34 by immunomagnetic positive selection. Methylcellulose cultureswere initiated with 1000 cells in complete methylcellulose media withouterythropoietin (Stem Cell Technologies, Vancuver, BC). Culture mediumwas later supplemented with 50 ng/mL rhEpo or CHEPO-IgG1 and 50 ng/ml ofkit ligand (KL), which acts as a stem cell factor (also called scf), andsynergizes in these assays with Epo to promote the formation ofarythroid colonies. After 12-14 days, colonies were enumerated andphenotyped on an inverted light microscope. The efficiency of CHEPO-IgG1in stimulating production of erythroid colonies from human bone marrowcells was measured and compared to the results from assays with rhEPO.Colony numbers from quadruplicate plates and repeated in two independentexperiments are presented in the following Table 7. TABLE 7 BFU-ECFU-GEMM CFU-GM Macrophage Experiment #1 KL/rhEpo 88 0 0 0 KL/CHEPO-IgGl82 0 1 0 Experiment #2 KL/rhEpo 104 0 0 0 KL/CHEPO-IgGl  92 0 0 0

[0342] C. Stimulation of bone marrow cells in liquid culture

[0343] The biological activity of CHEPO-IgG1was assessed in a thirdassay, measuring stimulation of bone marrow cells in liquid culture.Twenty thousand CD34+ cells isolated as described above were cultured inIMDM/10% FCS in the presence of CF, 50 ng/ml of kit ligand (KL) andeither 50 ng/mL rhEpo (Amgen) or CHEPO-IgG1. 7-10 inch plates of culturecells were counted by a hematocytometer and subsequently assayed forexpression of the erythroid cell surface markers CD36, CD71 andGlycophorin A. As shown in Table 8 and FIG. 5, CHEPO-IgG1 was asefficient as rhEPO in stimulating the formation of immature and matureerythroid cells in bone marrow liquid culture. TABLE 8 Condition Totalcellularity KL 2.50 + 05 KL/rhEPO 2.20E + 06   KL/CHEPO-IgGl 2.06 + 06

[0344]

1 52 1 2329 DNA Pan troglodytes misc_feature (1)...(2329) n = a, t, c org 1 ccccctggac agccgccctc tcctccaggc ccgtggggct ggccctgcac cgccgagctt 60cccgggatga gggcccccgg tgtggtcacc cggcgcgccc caggtcgctg agggaccccg 120gccaggcgcg gagatggggg tgcacggtga gtactcgcgg gctgggcgct cccgcccgcc 180cgggtccctg tttgagcggg gatttagcgc ccgggctatt ggccgggagg tggctgggtt 240caaggaccgg cgacttgtca aggaccccgg aagggggagg ggggtggggc agcctccacg 300tgccagcggg gacttggggg agtccttggg gatggcaaaa acctgacctg tgaaggggac 360acagtttggg ggttgagggg aagaaggttt gggggttctg ctgtgccagt ggagaggaag 420ctgataagct gataacctgg gcgctggagc caccacttat ctgccagagg gnnnntggta 480gctgggggtg gggtgtgcac acggcagcag gattgaatga aggccaggga ggcagcacct 540gagtgcttgc atggttgggg acaggaagga cgagctgggg cagagacgtg gggatgaagg 600aagctgtcct tccacagcca cccttctccc tccccgcctg actctcagcc tggctatctc 660ttctagaatg tcctgcctgg ctgtggcttc tcctgtccct gctgtcgctc cctctgggcc 720tcccagtcct gggcgcccca ccacgcctca tctgtgacag ccgagtcctg gagaggtacc 780tcttggaggc caaggaggcc gagaatatca cggtgagacc ccttccccag cacattccac 840agaactcacg ctcagggctt cagggaactc ctcccagatc caggaacctg gcacttggtt 900tggggtggag ttgggaagct agacactgcc cccctacata agaataagtc tggtggcccc 960aaaccatacc tggaaactag gcaaggagca aagccagcag atcctacggc ctgtgggcca 1020gggccagagc cttcagggac ccttgactcc ccgggctgtg tgcatttcag acgggctgtg 1080ccgaacactg cagcttgaat gagaatatca ctgtcccaga caccaaagtt aatttctatg 1140cctggaagag gatggaggtg agttcctttt tttttttttt tcctttcttt tggagaatct 1200catttgcgag cctgattttg gatgaaaggg agaatgatcg agggaaaggt aaaatggagc 1260agcagagatg aggctgcctg ggcgcagagg ctcacgtcta taatcccagg ctgagatggc 1320cgagatggga gaattgcttg agccctggag tttcagacca acctgggcag catagtgaga 1380tcccccatct ctacaaacat ttaaaaaaat tagtcaggtg aggtggtgca tggtggtagt 1440cccagatatt tggaaggctg aggcgggagg atcgcttgag cccaggaatt tgaggctgca 1500gtgagctgtg atcacaccac tgcactccag cctcagtgac agagtgaggc cctgtctcaa 1560aaaagaaaag aaaaaagaaa aataatgagg gctgtatgga atacattcat tattcattca 1620ctcactcatt cattcattca ttcattcnnn nnntcttatt gcatacctct gtttgctcag 1680cttggtgctt ggggctgctg aggggcagga gggagagggt ggcatgggtc agctgactcc 1740cagagtccac tccctgtagg tcaggcagca ggccgtagaa gtctggcagg gcctggccct 1800gctctcggaa gctgtcctgc ggggccaggc cctgttggtc aactcttccc agccgtggga 1860gcccctgcag ctgcatgtgg ataaagccgt cagtggcctt cgcagcctca ccactctgct 1920tcgggctctg ggagcccagg tgagtaggag cggacacttc tgcttgccct ttctgtaaga 1980aagggagaag ggtcttgcta aggagtacag gaactgtccg tattccttcc ccttctgtgg 2040cactgcagcg acctcctgtt ttctccttgg cagaaggaag ccatctcccc tccagatgcg 2100gcctcagctg ctccactccg aacaatcact gctgacactt tccgcaaact cttccgagtc 2160tactccaatt tcctccgggg aaagctgaag ctgtacacag gggaggcctg caggacaggg 2220gacagatgac caggtgtgtc cacctgggca tatccaccac ctccctcacc aacattgctt 2280gtgccacacc ctcccccgcc actcctgaac cccgtcgagg agctctcag 2329 2 193 PRT Pantroglodytes 2 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu LeuSer Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala ProPro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu GluAla Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys SerLeu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr AlaTrp Lys Arg 65 70 75 80 Met Glu Val Arg Gln Gln Ala Val Glu Val Trp GlnGly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu LeuVal Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val AspLys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg AlaLeu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala SerAla Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg LysLeu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys LeuTyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 3 585 DNA Pantroglodytes 3 atgggggtgc acgaatgtcc tgcctggctg tggcttctcc tgtccctgctgtcgctccct 60 ctgggcctcc cagtcctggg cgccccacca cgcctcatct gtgacagccgagtcctggag 120 aggtacctct tggaggccaa ggaggccgag aatatcacga cgggctgtgccgaacactgc 180 agcttgaatg agaatatcac tgtcccagac accaaagtta atttctatgcctggaagagg 240 atggaggtca ggcagcaggc cgtagaagtc tggcagggcc tggccctgctctcggaagct 300 gtcctgcggg gccaggccct gttggtcaac tcttcccagc cgtgggagcccctgcagctg 360 catgtggata aagccgtcag tggccttcgc agcctcacca ctctgcttcgggctctggga 420 gcccagaagg aagccatctc ccctccagat gcggcctcag ctgctccactccgaacaatc 480 actgctgaca ctttccgcaa actcttccga gtctactcca atttcctccggggaaagctg 540 aagctgtaca caggggaggc ctgcaggaca ggggacagat gacca 585 4193 PRT Homo sapiens 4 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp LeuLeu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu GlyAla Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr LeuLeu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu HisCys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn PheTyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Gly Gln Gln Ala Val Glu ValTrp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu LeuArg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp AlaAla Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr PheArg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys LeuLys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 5 193PRT Pan troglodytes 5 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp LeuLeu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu GlyAla Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr LeuLeu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu HisCys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn PheTyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Arg Gln Gln Ala Val Glu ValTrp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu LeuArg Ala Leu Gly Ala Lys Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp AlaAla Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr PheArg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys LeuLys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 6 4 PRTPan troglodytes 6 Met Glu Val Arg 1 7 4 PRT Pan troglodytes UNSURE 2, 4Xaa = unknown amino acid 7 Asn Xaa Ser Xaa 1 8 4 PRT Pan troglodytesUNSURE 2, 4 Xaa = unknown amino acid 8 Asn Xaa Thr Xaa 1 9 4 PRT Pantroglodytes 9 Glu Val Arg Gln 1 10 4 PRT Pan troglodytes 10 Val Arg GlnGln 1 11 4 PRT Pan troglodytes 11 Arg Gln Gln Ala 1 12 18 DNA Pantroglodytes 12 accgcgcccc ctggacag 18 13 25 DNA Pan troglodytes 13catccacttc tccggccaaa cttca 25 14 21 DNA Pan troglodytes 14 tttggccggagaagtggatg c 21 15 31 DNA Pan troglodytes 15 tcactcactc actcattcattcattcattc a 31 16 29 DNA Pan troglodytes 16 gttgaatgaa tgattgaatgaatgagtga 29 17 23 DNA Pan troglodytes 17 gcactggagt gtccatggga cag 2318 166 PRT Pan troglodytes UNSURE 55, 57 Xaa = unknown amino acid 18 AlaPro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45Tyr Ala Trp Lys Arg Asn Xaa Ser Xaa Gln Gln Ala Val Glu Val Trp 50 55 60Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 7580 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 9095 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe ArgVal 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr GlyGlu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 19 166 PRT Pantroglodytes UNSURE 55, 57 Xaa = unknown amino acid 19 Ala Pro Pro ArgLeu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu AlaLys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser LeuAsn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala TrpLys Arg Asn Xaa Ser Xaa Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly LeuAla Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu ValAsn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys AlaVal Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 GlyAla Lys Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145150 155 160 Cys Arg Thr Gly Asp Arg 165 20 166 PRT Pan troglodytesUNSURE 55, 57 Xaa = unknown amino acid 20 Ala Pro Pro Arg Leu Ile CysAsp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu AlaGlu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu AsnIle Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg AsnXaa Thr Xaa Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu LeuSer Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser SerGln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser GlyLeu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln LysGlu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu ArgThr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr SerAsn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160Cys Arg Thr Gly Asp Arg 165 21 166 PRT Pan troglodytes UNSURE 55, 57 Xaa= unknown amino acid 21 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val LeuGlu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr ThrGly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro AspThr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Asn Xaa Thr Xaa Gln GlnAla Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val LeuArg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu ProLeu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu ThrThr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Lys Lys Glu Ala Ile Ser ProPro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala AspThr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg GlyLys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly AspArg 165 22 166 PRT Pan troglodytes UNSURE 56, 58 Xaa = unknown aminoacid 22 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala GluHis 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val AsnPhe 35 40 45 Tyr Ala Trp Lys Arg Met Asn Xaa Ser Xaa Gln Ala Val Glu ValTrp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu ArgAla Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala AlaSer Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg LysLeu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys LeuTyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 23 166PRT Pan troglodytes UNSURE 56, 58 Xaa = unknown amino acid 23 Ala ProPro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 LeuGlu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 CysSer Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 TyrAla Trp Lys Arg Met Asn Xaa Ser Xaa Gln Ala Val Glu Val Trp 50 55 60 GlnGly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105110 Gly Ala Lys Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly GluAla 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 24 166 PRT Pantroglodytes UNSURE 56, 58 Xaa = unknown amino acid 24 Ala Pro Pro ArgLeu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu AlaLys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser LeuAsn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala TrpLys Arg Met Asn Xaa Thr Xaa Gln Ala Val Glu Val Trp 50 55 60 Gln Gly LeuAla Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu ValAsn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys AlaVal Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 GlyAla Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145150 155 160 Cys Arg Thr Gly Asp Arg 165 25 166 PRT Pan troglodytesUNSURE 56, 58 Xaa = unknown amino acid 25 Ala Pro Pro Arg Leu Ile CysAsp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu AlaGlu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu AsnIle Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg MetAsn Xaa Thr Xaa Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu LeuSer Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser SerGln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser GlyLeu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Lys LysGlu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu ArgThr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr SerAsn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160Cys Arg Thr Gly Asp Arg 165 26 166 PRT Pan troglodytes UNSURE 57, 59 Xaa= unknown amino acid 26 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val LeuGlu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr ThrGly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro AspThr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Asn Xaa Ser XaaAla Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val LeuArg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu ProLeu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu ThrThr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser ProPro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala AspThr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg GlyLys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly AspArg 165 27 166 PRT Pan troglodytes UNSURE 57, 59 Xaa = unknown aminoacid 27 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala GluHis 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val AsnPhe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Asn Xaa Ser Xaa Ala Val Glu ValTrp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu ArgAla Leu 100 105 110 Gly Ala Lys Lys Glu Ala Ile Ser Pro Pro Asp Ala AlaSer Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg LysLeu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys LeuTyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 28 166PRT Pan troglodytes UNSURE 57, 59 Xaa = unknown amino acid 28 Ala ProPro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 LeuGlu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 CysSer Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 TyrAla Trp Lys Arg Met Glu Asn Xaa Thr Xaa Ala Val Glu Val Trp 50 55 60 GlnGly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly GluAla 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 29 166 PRT Pantroglodytes UNSURE 57, 59 Xaa = unknown amino acid 29 Ala Pro Pro ArgLeu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu AlaLys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser LeuAsn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala TrpLys Arg Met Glu Asn Xaa Thr Xaa Ala Val Glu Val Trp 50 55 60 Gln Gly LeuAla Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu ValAsn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys AlaVal Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 GlyAla Lys Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145150 155 160 Cys Arg Thr Gly Asp Arg 165 30 166 PRT Pan troglodytesUNSURE 58, 60 Xaa = unknown amino acid 30 Ala Pro Pro Arg Leu Ile CysAsp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu AlaGlu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu AsnIle Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg MetGlu Val Asn Xaa Ser Xaa Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu LeuSer Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser SerGln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser GlyLeu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Gln LysGlu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu ArgThr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr SerAsn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160Cys Arg Thr Gly Asp Arg 165 31 166 PRT Pan troglodytes UNSURE 58, 60 Xaa= unknown amino acid 31 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val LeuGlu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr ThrGly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro AspThr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Asn Xaa SerXaa Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val LeuArg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu ProLeu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu ThrThr Leu Leu Arg Ala Leu 100 105 110 Gly Ala Lys Lys Glu Ala Ile Ser ProPro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala AspThr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg GlyLys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly AspArg 165 32 166 PRT Pan troglodytes UNSURE 58, 60 Xaa = unknown aminoacid 32 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala GluHis 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val AsnPhe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Asn Xaa Thr Xaa Val Glu ValTrp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu ArgAla Leu 100 105 110 Gly Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala AlaSer Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg LysLeu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys LeuTyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 33 166PRT Pan troglodytes UNSURE 58, 60 Xaa = unknown amino acid 33 Ala ProPro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 LeuGlu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 CysSer Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 TyrAla Trp Lys Arg Met Glu Val Asn Xaa Thr Xaa Val Glu Val Trp 50 55 60 GlnGly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105110 Gly Ala Lys Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly GluAla 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 34 193 PRT Pantroglodytes UNSURE 82, 84 Xaa = unknown amino acid 34 Met Gly Val HisGlu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser LeuPro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys AspSer Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu AsnIle Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile ThrVal Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Asn XaaSer Xaa Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu SerGlu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 GlnPro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn PheLeu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg ThrGly Asp 180 185 190 Arg 35 193 PRT Pan troglodytes UNSURE 82, 84 Xaa =unknown amino acid 35 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp LeuLeu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu GlyAla Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr LeuLeu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu HisCys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn PheTyr Ala Trp Lys Arg 65 70 75 80 Asn Xaa Ser Xaa Gln Gln Ala Val Glu ValTrp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu LeuArg Ala Leu Gly Ala Lys Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp AlaAla Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr PheArg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys LeuLys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 36 193PRT Pan troglodytes UNSURE 82, 84 Xaa = unknown amino acid 36 Met GlyVal His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 LeuSer Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 IleCys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 AlaGlu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 AsnIle Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80Asn Xaa Thr Xaa Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg ThrIle 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr SerAsn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala CysArg Thr Gly Asp 180 185 190 Arg 37 193 PRT Pan troglodytes UNSURE 82, 84Xaa = unknown amino acid 37 Met Gly Val His Glu Cys Pro Ala Trp Leu TrpLeu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val LeuGly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg TyrLeu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala GluHis Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val AsnPhe Tyr Ala Trp Lys Arg 65 70 75 80 Asn Xaa Thr Xaa Gln Gln Ala Val GluVal Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly GlnAla Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln LeuHis Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr LeuLeu Arg Ala Leu Gly Ala Lys Lys Glu 130 135 140 Ala Ile Ser Pro Pro AspAla Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp ThrPhe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly LysLeu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 38193 PRT Pan troglodytes UNSURE 83, 85 Xaa = unknown amino acid 38 MetGly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 7580 Met Asn Xaa Ser Xaa Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 9095 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln LysGlu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu ArgThr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val TyrSer Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu AlaCys Arg Thr Gly Asp 180 185 190 Arg 39 193 PRT Pan troglodytes UNSURE83, 85 Xaa = unknown amino acid 39 Met Gly Val His Glu Cys Pro Ala TrpLeu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu ProVal Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu GluArg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly CysAla Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr LysVal Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Asn Xaa Ser Xaa Gln AlaVal Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu ArgGly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro LeuGln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu ThrThr Leu Leu Arg Ala Leu Gly Ala Lys Lys Glu 130 135 140 Ala Ile Ser ProPro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr AlaAsp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 ArgGly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190Arg 40 193 PRT Pan troglodytes UNSURE 83, 85 Xaa = unknown amino acid 40Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 1015 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 2530 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 4045 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 5560 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 7075 80 Met Asn Xaa Thr Xaa Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 8590 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val SerGly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala GlnLys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro LeuArg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg ValTyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly GluAla Cys Arg Thr Gly Asp 180 185 190 Arg 41 193 PRT Pan troglodytesUNSURE 83, 85 Xaa = unknown amino acid 41 Met Gly Val His Glu Cys ProAla Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu GlyLeu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg ValLeu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr ThrGly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro AspThr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Asn Xaa Thr XaaGln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala ValLeu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp GluPro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg SerLeu Thr Thr Leu Leu Arg Ala Leu Gly Ala Lys Lys Glu 130 135 140 Ala IleSer Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180185 190 Arg 42 193 PRT Pan troglodytes UNSURE 84, 86 Xaa = unknown aminoacid 42 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro ArgLeu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala LysGlu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu AsnGlu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp LysArg 65 70 75 80 Met Glu Asn Xaa Ser Xaa Ala Val Glu Val Trp Gln Gly LeuAla Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val AsnSer Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys AlaVal Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu GlyAla Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala AlaPro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu PheArg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr ThrGly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 43 193 PRT Pantroglodytes UNSURE 84, 86 Xaa = unknown amino acid 43 Met Gly Val HisGlu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser LeuPro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys AspSer Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu AsnIle Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile ThrVal Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met GluAsn Xaa Ser Xaa Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu SerGlu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 GlnPro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Lys Lys Glu 130 135140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn PheLeu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg ThrGly Asp 180 185 190 Arg 44 193 PRT Pan troglodytes UNSURE 84, 86 Xaa =unknown amino acid 44 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp LeuLeu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu GlyAla Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr LeuLeu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu HisCys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val Asn PheTyr Ala Trp Lys Arg 65 70 75 80 Met Glu Asn Xaa Thr Xaa Ala Val Glu ValTrp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly Gln AlaLeu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu HisVal Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu LeuArg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp AlaAla Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr PheArg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys LeuLys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 45 193PRT Pan troglodytes UNSURE 84, 86 Xaa = unknown amino acid 45 Met GlyVal His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15 LeuSer Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30 IleCys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 AlaGlu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 AsnIle Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80Met Glu Asn Xaa Thr Xaa Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100 105110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly 115120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Lys Lys Glu130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg ThrIle 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr SerAsn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala CysArg Thr Gly Asp 180 185 190 Arg 46 193 PRT Pan troglodytes UNSURE 85, 87Xaa = unknown amino acid 46 Met Gly Val His Glu Cys Pro Ala Trp Leu TrpLeu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val LeuGly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu Arg TyrLeu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly Cys Ala GluHis Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr Lys Val AsnPhe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Asn Xaa Ser Xaa Val GluVal Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu Arg Gly GlnAla Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro Leu Gln LeuHis Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu Thr Thr LeuLeu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro AspAla Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp ThrPhe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly LysLeu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 47193 PRT Pan troglodytes UNSURE 85, 87 Xaa = unknown amino acid 47 MetGly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 10 15Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 25 30Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 7580 Met Glu Val Asn Xaa Ser Xaa Val Glu Val Trp Gln Gly Leu Ala Leu 85 9095 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Lys LysGlu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu ArgThr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val TyrSer Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu AlaCys Arg Thr Gly Asp 180 185 190 Arg 48 193 PRT Pan troglodytes UNSURE85, 87 Xaa = unknown amino acid 48 Met Gly Val His Glu Cys Pro Ala TrpLeu Trp Leu Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu ProVal Leu Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu GluArg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr Gly CysAla Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val Pro Asp Thr LysVal Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu Val Asn Xaa Thr XaaVal Glu Val Trp Gln Gly Leu Ala Leu 85 90 95 Leu Ser Glu Ala Val Leu ArgGly Gln Ala Leu Leu Val Asn Ser Ser 100 105 110 Gln Pro Trp Glu Pro LeuGln Leu His Val Asp Lys Ala Val Ser Gly 115 120 125 Leu Arg Ser Leu ThrThr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu 130 135 140 Ala Ile Ser ProPro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile 145 150 155 160 Thr AlaAsp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 ArgGly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190Arg 49 193 PRT Pan troglodytes UNSURE 85, 87 Xaa = unknown amino acid 49Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu 1 5 1015 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu 20 2530 Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu 35 4045 Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 5560 Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 7075 80 Met Glu Val Asn Xaa Thr Xaa Val Glu Val Trp Gln Gly Leu Ala Leu 8590 95 Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser100 105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val SerGly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala LysLys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro LeuArg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg ValTyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu Tyr Thr Gly GluAla Cys Arg Thr Gly Asp 180 185 190 Arg 50 42 DNA Synthetic 50cggaattcat gggggtgcac gaatgtcctg cctggctgtg gc 42 51 32 DNA Synthetic 51gctctagact catctgtccc ctgtcctgca gg 32 52 35 DNA Synthetic 52 gtccgggtgacccctctgtc ccctgtcctg caggc 35

What is claimed is:
 1. An isolated nucleic acid molecule having at leastabout 80% nucleic acid sequence identity to (a) a DNA molecule encodinga CHEPO polypeptide comprising the sequence of amino acid residues fromabout 1 or about 28 to about 193 of FIG. 2 (SEQ ID NO:2), or (b) thecomplement of the DNA molecule of (a).
 2. The isolated nucleic acidmolecule of claim 1 comprising nucleotides 1 or about 82 to about 579 ofFIG. 2 (SEQ ID NO:3).
 3. The isolated nucleic acid molecule of claim 1comprising the nucleotide sequence of FIG. 2 (SEQ ID NO:3).
 4. Theisolated nucleic acid molecule of claim 1 comprising a nucleotidesequence that encodes the sequence of amino acid residues from about 1or about 28 to about 193 of FIG. 2 (SEQ ID NO:2).
 5. An isolated nucleicacid molecule encoding a CHEPO polypeptide comprising DNA thathybridizes to the complement of the nucleic acid sequence that encodesamino acids 1 or about 28 to about 193 of FIG. 2 (SEQ ID NO:2).
 6. Theisolated nucleic acid molecule of claim 5, wherein the nucleic acid thatencodes amino acids 1 or about 28 to about 193 of FIG. 2 (SEQ ID NO:2)comprises nucleotides 1 or about 82 to about 579 of FIG. 2 (SEQ IDNO:3).
 7. The isolated nucleic acid molecule of claim 5, wherein thehybridization occurs under stringent hybridization and wash conditions.8. An isolated nucleic acid molecule comprising (a) DNA encoding apolypeptide scoring at least 80% positives when compared to the sequenceof amino acid residues of from 1 or about 28 to about 193 of FIG. 2 (SEQID NO:2), or (b) the complement of the DNA of (a).
 9. A vectorcomprising the nucleic acid molecule of claim
 1. 10. The vector of claim9, wherein said nucleic acid molecule is operably linked to controlsequences recognized by a host cell transformed with the vector.
 11. Ahost cell comprising the vector of claim
 9. 12. The host cell of claim11, wherein said cell is a CHO cell.
 13. The host cell of claim 11,wherein said cell is an E. coli.
 14. The host cell of claim 11, whereinsaid cell is a yeast cell.
 15. A process for producing a CHEPOpolypeptide comprising culturing the host cell of claim 11 underconditions suitable for expression of said CHEPO polypeptide andrecovering said CHEPO polypeptide from the cell culture.
 16. An isolatedCHEPO polypeptide comprising an amino acid sequence comprising at leastabout 80% sequence identity to the sequence of amino acid residues fromabout 1 or about 28 to about 193 of FIG. 2 (SEQ ID NO:2).
 17. Theisolated CHEPO polypeptide of claim 16 comprising amino acid residues 1or about 28 to about 193 of FIG. 2 (SEQ ID NO:2).
 18. An isolated CHEPOpolypeptide scoring at least 80% positives when compared to the sequenceof amino acid residues from 1 or about 28 to about 193 of FIG. 2 (SEQ IDNO:2).
 19. An isolated CHEPO polypeptide comprising the sequence ofamino acid residues from 1 or about 28 to about 193 of FIG. 2 (SEQ IDNO:2), or a fragment thereof sufficient to provide a binding site for ananti-CHEPO antibody.
 20. An isolated polypeptide produced by (I)hybridizing a test DNA molecule under stringent conditions with (a) aDNA molecule encoding a CHEPO polypeptide comprising the sequence ofamino acid residues from 1 or about 28 to about 193 of FIG. 2 (SEQ IDNO:2), or (b) the complement of the DNA molecule of (a), (ii) culturinga host cell comprising said test DNA molecule under conditions suitablefor the expression of said polypeptide, and (iii) recovering saidpolypeptide from the cell culture.
 21. The isolated polypeptide of claim20, wherein said test DNA has at least about 80% sequence identity to(a) or (b).
 22. A chimeric molecule comprising a CHEPO polypeptide fusedto a heterologous amino acid sequence.
 23. The chimeric molecule ofclaim 22, wherein said heterologous amino acid sequence is an epitopetag sequence.
 24. The chimeric molecule of claim 22, wherein saidheterologous amino acid sequence is a Fc region of an immunoglobulin.25. An antibody which specifically binds to a CHEPO polypeptide.
 26. Theantibody of claim 24, wherein said antibody is a monoclonal antibody.27. The antibody of claim 24, wherein said antibody is a humanizedantibody.
 28. An agonist to a CHEPO polypeptide.
 29. An antagonist to aCHEPO polypeptide.
 30. A composition of matter comprising (a) a CHEPOpolypeptide, (b) an agonist to a CHEPO polypeptide, (c) an antagonist toa CHEPO polypeptide, or (d) an anti-CHEPO antibody in admixture with apharmaceutically acceptable carrier.
 31. A method of inducingerythropoiesis in a mammal, said method comprising administering to saidmammal an effective amount of a CHEPO polypeptide or an agonist thereto,wherein erythropoiesis in said mammal is induced.
 32. A method ofinhibiting erythropoiesis in a mammal, said method comprisingadministering to said mammal an effective amount of an antagonist to aCHEPO polypeptide, wherein erythropoiesis in said mammal is inhibited.33. The method according to claim 31, wherein said antagonist is ananti-CHEPO antibody.
 34. A CHEPO polypeptide comprising an amino acidsequence selected from the group consisting of:APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRNXSXQQAVEVW (SEQ IDNO:18); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRNXSXQQAVEVW (SEQ IDNO:19); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRNXTXQQAVEVW (SEQ IDNO:20); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRNXTXQQAVEVW (SEQ IDNO:21); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMNXSXQAVEV (SEQ IDNO:22); WQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMNXSXQAVEV (SEQ IDNO:23); WQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMNXTXQAVEV (SEQ IDNO:24); WQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMNXTXQAVEV (SEQ IDNO:25); WQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMENXSXAVEVW (SEQ IDNO:26); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMENXSXAVEVW (SEQ IDNO:27); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMENXTXAVEVW (SEQ IDNO:28); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMENXTXAVEVW (SEQ IDNO:29); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVNXSXVEVW (SEQ IDNO:30); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVNXSXVEVW (SEQ IDNO:31); QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVNXTXVEVW (SEQ IDNO:32); andQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVNXTXVEVW (SEQ IDNO:33) QGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR, wherein X is any amino acidexcept for proline.


35. The CHEPO polypeptide according to claim 34, which comprises anamino acid sequence selected from the group consisting of:MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:34); NITVPDTKVNFYAWKRNXSXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:35); NITVPDTKVNFYAWKRNXSXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:36); NITVPDTKVNFYAWKRNXTXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:37); NITVPDTKVNFYAWKRNXTXQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:38); NITVPDTKVNFYAWKRMNXSXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:39); NITVPDTKVNFYAWKRMNXSXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:40); NITVPDTKVNFYAWKRMNXTXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:41); NITVPDTKVNFYAWKRMNXTXQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:42); NITVPDTKVNFYAWKRMENXSXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:43); NITVPDTKVNFYAWKRMENXSXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:44); NITVPDTKVNFYAWKRMENXTXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:45); NITVPDTKVNFYAWKRMENXTXAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:46); NITVPDTKVNFYAWKRMEVNXSXVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:47); NITVPDTKVNFYAWKRMEVNXSXVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTG DRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:48); andNITVPDTKVNFYAWKRMEVNXTXVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDRMGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNE (SEQ IDNO:49) NITVPDTKVNFYAWKRMEVNXTXVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAKKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRT GDR,wherein X is any amino acid except for proline.


36. A chimeric molecule comprising a CHEPO polypeptide of claim 34 or 35fused to a heterologous amino acid sequence.
 37. The chimeric moleculeof claim 36, wherein said heterologous amino acid sequence is an epitopetag sequence.
 38. The chimeric molecule of claim 36, wherein saidheterologous amino acid sequence is an immunoglobulin constant domainsequence.
 39. The chimeric molecule of claim 38, wherein said constantdomain sequence is a Fc region of an immunoglobulin.
 40. The chimericmolecule of claim 39 wherein said immunoglobulin is an IgG.
 41. Thechimeric molecule of claim 40 wherein said IgG is IgG1.
 42. A method ofstimulating the proliferation of cells expressing EPO receptor, saidmethod comprising contacting said cells with an effective amount of aCHEPO polypeptide, wherein the proliferation of said cells isstimulated.
 43. The method of claim 42, wherein said CHEPO polypeptideis a CHEPO immunoadhesin.
 44. The method of claim 42, wherein said cellsare of hematopoietic origin.